Technical Field
[0001] The present invention relates to a high-shrinkage artificial fibroin spun yarn and
a method for manufacturing the same, and an artificial fibroin spun yarn and a method
for shrinking the same.
Background Art
[0002] In the related art, spun yarns made of synthetic fibers, natural fibers, or the like
have been used for various knitted fabric materials. Thus, when knitted fabrics are
manufactured, spun yarns that can satisfy requirements for the knitted fabrics are
selected. For example, when clothes, bedclothes, and the like having an expensive
feeling and being luxurious are manufactured, silk spun yarns and the like are selected
as materials thereof.
[0003] In addition, depending on the properties required for knitted fabrics, spun yarns
as materials therefor may be subjected to predetermined processing. For example, in
clothes, bedclothes and the like, when higher flexibility and heat retention are desired
in addition to an expensive feeling and a luxurious feeling, silk spun yams that have
been subjected to shrinkage processing such that bulkiness may be increased may be
used.
[0004] Regarding a silk shrinkage method, for example, so-called salt shrinkage processing
in which silk is immersed in an aqueous solution in which inorganic salts such as
calcium nitrate and calcium chloride are dissolved in a high concentration (salt shrinkage
solution) and shrunk is known (for example, Patent Literature 1).
[0005] On the other hand, for example, synthetic fibers such as polyester fibers, polyamide
fibers and acrylic fibers which are generally used for clothes, bedclothes, and the
like may be brought into contact with boiling water, and thus a shrinkage rate of
40% or more may be realized (Patent Literature 2).
Citation List
Patent Literature
[0006]
[Patent Literature 1] Japanese Unexamined Patent Publication No. 2001-64866
[Patent Literature 2] Japanese Unexamined Patent Publication No. 2009-121003
Summary of Invention
Technical Problem
[0007] However, silk has a very small shrinkage rate when it is simply brought into contact
with water. In addition, the shrinkage method described in Patent Literature 2 has
a high risk because boiling water with a high temperature is handled.
[0008] An objective of the present invention is to provide a high-shrinkage artificial fibroin
spun yarn which has a sufficiently high shrinkage rate and an excellent tactile feel
and flexibility, and also can be manufactured safely and a method for manufacturing
the same. Another objective of the present invention is to provide an artificial fibroin
spun yarn which allows a high-shrinkage artificial fibroin spun yarn to be manufactured
at a sufficiently high shrinkage rate and safely, and a method for shrinking the same.
Solution to Problem
[0009] For example, the present invention relates to the following inventions.
- [1] A high-shrinkage artificial fibroin spun yarn which is a shrunk artificial fibroin
spun yarn including a modified fibroin and having a shrinkage rate of greater than
1% defined by the following Formula I:
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0001)
- [2] The high-shrinkage artificial fibroin spun yarn according to [1],
wherein the modified fibroin is a modified spider silk fibroin.
- [3] The high-shrinkage artificial fibroin spun yarn according to [1] or [2], which
is shrunk when brought into contact with water having a temperature lower than a boiling
point.
- [4] The high-shrinkage artificial fibroin spun yarn according to [3],
wherein the temperature of the water is 10 to 90°C.
- [5] The high-shrinkage artificial fibroin spun yarn according to [3] or [4], which
is additionally shrunk when drying is performed after contact with the water.
- [6] A method for manufacturing a high-shrinkage artificial fibroin spun yarn, including:
a shrinkage process in which an artificial fibroin spun yarn including a modified
fibroin is brought into contact with water having a temperature lower than a boiling
point,
wherein a shrinkage rate defined by the following Formula I is greater than 1%:
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0002)
- [7] The method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to [6],
wherein the modified fibroin is a modified spider silk fibroin.
- [8] The method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to [6] or [7],
wherein the temperature of the water is 10 to 90°C.
- [9] The method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [8],
wherein the shrinkage process further includes drying of the artificial fibroin spun
yarn after it is brought into contact with the water.
- [10] A method for shrinking an artificial fibroin spun yarn, including:
a shrinkage process in which an artificial fibroin spun yarn including a modified
fibroin is brought into contact with water having a temperature lower than a boiling
point,
wherein a shrinkage rate defined by the following Formula I is greater than 1%:
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0003)
- [11] The method for shrinking an artificial fibroin spun yarn according to [10], wherein
the modified fibroin is a modified spider silk fibroin.
- [12] The method for shrinking an artificial fibroin spun yarn according to [10] or
[11],
wherein the temperature of the water is 10 to 90°C.
- [13] The method for shrinking an artificial fibroin spun yarn according to any one
of [10] to [12],
wherein the shrinkage process further includes drying of the artificial fibroin spun
yarn after it is brought into contact with the water.
- [14] An artificial fibroin spun yarn including a modified fibroin and having a shrinkage
rate of greater than 1% defined by the following Formula II:
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0004)
- [15] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m, and
wherein the domain sequence includes an amino acid sequence with a reduced content
of the (A)n motif, which corresponds to deletion of at least one or more (A)n motifs, as compared with a naturally derived fibroin:
[in Formula 1, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [16] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [15], wherein
the domain sequence includes an amino acid sequence corresponding to a case in which
at least one (A)n motif for every one to three (A)n motifs from the N-terminal side to the C-terminal side is deleted, as compared with
a naturally derived fibroin.
- [17] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [15], wherein
the domain sequence includes an amino acid sequence corresponding to a case in which,
from the N-terminal side to the C-terminal side, at least deletion of two consecutive
(A)n motifs and deletion of one (A)n motif are repeated in that order, as compared with a naturally derived fibroin.
- [18] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m, and
wherein, when the numbers of amino acid residues of REP of adjacent two [(A)n motif-REP] units are sequentially compared from the N-terminal side to the C-terminal
side, the maximum value of the total value obtained by adding the number of amino
acid residues of the adjacent two [(A)n motif-REP] units in which a ratio of the number of amino acid residues of the other
REP with respect to 1 set for the number of amino acid residues of one REP with a
smaller number of amino acid residues is 1.8 to 11.3 is set as x, and the total number
of amino acid residues of the domain sequence is set as y, x/y is 50% or more:
[in Formula 1, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [19] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m, and
wherein the domain sequence includes an amino acid sequence with a reduced content
of glycine residues, which corresponds to substitution of at least one or more glycine
residues in REP with other amino acid residues, as compared with a naturally derived
fibroin:
[in Formula 1, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [20] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [19],
wherein the domain sequence includes an amino acid sequence corresponding to a case
in which, in at least one motif sequence selected from among GGX and GPGXX (where,
X represents an amino acid residue other than glycine) in REP, one glycine residue
in at least one or more motif sequences is substituted with another amino acid residue,
as compared with a naturally derived fibroin.
- [21] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [20],
wherein a proportion of the motif sequences in which a glycine residue is substituted
with another amino acid residue with respect to all motif sequences is 10% or more.
- [22] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m, and
wherein, when the total number of amino acid residues of amino acid sequences composed
of XGX (where, X represents an amino acid residue other than glycine) contained in
all REPs in the sequence obtained by removing a sequence from the (A)n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as z, and the total number of amino acid residues
in the sequence obtained by removing a sequence from the (A)n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as w, z/w is 50.9% or more:
[in Formula 1, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [23] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [19]
to [22],
wherein the modified fibroin includes an amino acid sequence corresponding to a case
in which one or more glycine residues in REP are substituted with other amino acid
residues and additionally, one or more amino acid residues are substituted, deleted,
inserted and/or added, as compared with a naturally derived fibroin.
- [24] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m, and
wherein the domain sequence includes an amino acid sequence including a region having
a locally large hydrophobicity index, which corresponds to substitution of one or
more amino acid residues in REP with an amino acid residue having a large hydrophobicity
index and/or insertion of one or more amino acid residues having a large hydrophobicity
index into REP, as compared with a naturally derived fibroin:
[in Formula 1, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [25] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [24],
wherein the region having a locally large hydrophobicity index is composed of 2 to
4 consecutive amino acid residues.
- [26] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [24] or [25],
wherein the amino acid residue having a large hydrophobicity index is selected from
among isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine
(M) and alanine (A).
- [27] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m, and
wherein, when the total number of amino acid residues included in a region in which
the average value of the hydrophobicity index of 4 consecutive amino acid residues
is 2.6 or more in all REPs included in the sequence obtained by removing a sequence
from the (A)n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as p, and the total number of amino acid residues
included in the sequence obtained by removing a sequence from the (A)n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as q, p/q is 6.2% or more:
[in Formula 1, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 83% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [28] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [24]
to [27],
wherein the modified fibroin includes an amino acid sequence corresponding to a case
in which one or more amino acid residues in REP are substituted with an amino acid
residue having a large hydrophobicity index and/or one or more amino acid residues
having a large hydrophobicity index are inserted into REP, and additionally, one or
more amino acid residues are substituted, deleted, inserted and/or added, as compared
with a naturally derived fibroin.
- [29] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin includes a domain sequence represented by Formula 1:
[(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, and
wherein the domain sequence includes an amino acid sequence with a reduced content
of glutamine residues, which corresponds to deletion of one or more glutamine residues
in REP or substitution with other amino acid residues, as compared with a naturally
derived fibroin:
[in Formula 1 and in Formula 2, the (A)n motif represents an amino acid sequence composed of 2 to 27 amino acid residues and
the number of alanine residues with respect to the total number of amino acid residues
in the (A)n motif is 80% or more; REP represents an amino acid sequence composed of 10 to 200
amino acid residues; m represents an integer of 2 to 300; a plurality of (A)n motifs may have the same amino acid sequence or different amino acid sequences; and
a plurality of REPs may have the same amino acid sequence or different amino acid
sequences].
- [30] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [29],
wherein the modified fibroin includes a GPGXX (where, X represents an amino acid residue
other than a glycine residue) motif in REP, and the content of the GPGXX motif is
10% or more.
- [31] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to [29] or [30],
wherein the modified fibroin has a glutamine residue content of 9% or less.
- [32] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [29]
to [31],
wherein the other amino acid residue is an amino acid residue selected from the group
consisting of isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine
(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan
(W), tyrosine (Y), proline (P) and histidine (H).
- [33] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [29]
to [32],
wherein, in the modified fibroin, the hydrophobicity of REP is -0.8 or more.
- [34] The high-shrinkage artificial fibroin spun yarn, the method for manufacturing
a high-shrinkage artificial fibroin spun yarn, the method for shrinking an artificial
fibroin spun yarn, or the artificial fibroin spun yarn according to any one of [29]
to [33],
wherein the modified fibroin includes an amino acid sequence corresponding to a case
in which one or more glutamine residues in REP are deleted or substituted with other
amino acid residues and additionally one or more amino acid residues are substituted,
deleted, inserted and/or added, as compared with a naturally derived fibroin.
- [35] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin has a limit oxygen index (LOI) value of 26.0 or more.
- [36] The high-shrinkage artificial fibroin spun yarn according to any one of [1] to
[5], the method for manufacturing a high-shrinkage artificial fibroin spun yarn according
to any one of [6] to [9], the method for shrinking an artificial fibroin spun yarn
according to any one of [10] to [13], or the artificial fibroin spun yarn according
to [14],
wherein the modified fibroin has a maximum hygroscopic heat generation of greater
than 0.025°C/g determined according to the following Formula A:
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0005)
[in Formula A, a low humidity environment means an environment of a temperature of
20°C and a relative humidity of 40%, and a high humidity environment means an environment
of a temperature of 20°C and a relative humidity of 90%].
Effects of Invention
[0010] According to the present invention, it is possible to provide a high-shrinkage artificial
fibroin spun yarn which has a sufficiently high shrinkage rate and an excellent tactile
feel and flexibility, and also can be manufactured safely and a method for manufacturing
the same. According to the present invention, it is also possible to provide an artificial
fibroin spun yarn which allows a high-shrinkage artificial fibroin spun yarn to be
manufactured at a sufficiently high shrinkage rate and safely, and a method for shrinking
the same.
Brief Description of Drawings
[0011]
FIG. 1 is a schematic view showing a domain sequence of a modified fibroin according
to one embodiment.
FIG. 2 is a diagram showing a distribution of values of z/w (%) of naturally derived
fibroin.
FIG. 3 is a diagram showing a distribution of values of x/y (%) of naturally derived
fibroin.
FIG. 4 is a schematic view showing a domain sequence of a modified fibroin according
to one embodiment.
FIG. 5 is a schematic view showing a domain sequence of a modified fibroin according
to one embodiment.
FIG. 6 is an explanatory diagram schematically showing an example of a spinning device
for manufacturing modified fibroin fibers (filaments).
FIG. 7 is a graph showing an example of results of a hygroscopic heat generation test.
Description of Embodiments
[0012] Forms for implementing the present invention will be described below in detail. However,
the present invention is not limited to the following embodiments.
[0013] [High-shrinkage artificial fibroin spun yarn] A high-shrinkage artificial fibroin
spun yarn according to the present embodiment is a shrunk artificial fibroin fiber
containing a modified fibroin. The high-shrinkage artificial fibroin spun yarn according
to the present embodiment has a shrinkage rate of greater than 1% defined in the following
Formula I.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0006)
<Modified fibroin>
[0014] The modified fibroin according to the present embodiment is a protein including a
domain sequence represented by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif. In the modified fibroin, an amino acid sequence (N-terminal sequence and C-terminal
sequence) may be additionally added to one or both of the N-terminal side and the
C-terminal side of the domain sequence. Although not limited to this, the N-terminal
sequence and the C-terminal sequence are typically regions having no repeat of an
amino acid motif characteristic of fibroin and are composed of amino acids of about
100 residues.
[0015] "Modified fibroin" in this specification means an artificially manufactured fibroin
(artificial fibroin). The modified fibroin may be a fibroin whose domain sequence
is different from an amino acid sequence of a naturally derived fibroin, or may a
fibroin having an amino acid sequence the same as that of a naturally derived fibroin.
"Naturally derived fibroin" in this specification is also a protein including a domain
sequence represented by Formula 1: [(A)
n motif-REP]
m, or Formula 2: [(A)
n motif-REP]
m-(A)
n motif.
[0016] "Modified fibroin" may be a fibroin in which an amino acid sequence of a naturally
derived fibroin is used without change, a fibroin whose amino acid sequence is modified
from the amino acid sequence of a naturally derived fibroin (for example, a fibroin
whose amino acid sequence is modified by modifying a cloned gene sequence of a naturally
derived fibroin), or a fibroin that is artificially designed and synthesized regardless
of a naturally derived fibroin (for example, a fibroin having a desired amino acid
sequence obtained by chemically synthesizing nucleic acids that encode a designed
amino acid sequence). Here, regarding the modified fibroin according to the present
embodiment, a modified spider silk fibroin is preferably used because it has excellent
heat retention, hygroscopic heat generation characteristics and/or flame retardancy.
[0017] "Domain sequence" in this specification refers to an amino acid sequence that generates
a crystalline region specific to a fibroin (typically, a region corresponding to the
(A)
n motif of an amino acid sequence) and a non-crystalline region (typically, a region
corresponding to an REP of an amino acid sequence) and an amino acid sequence represented
by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif. Here, the (A)
n motif represents an amino acid sequence mainly containing alanine residues, and the
number of amino acid residues is 2 to 27. The number of amino acid residues in the
(A)
n motif is an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to
16, or 10 to 16. In addition, a ratio of the number of alanine residues with respect
to the total number of amino acid residues in the (A)
n motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85%
or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed
of only alanine residues). A plurality of (A)
n motifs present in the domain sequence may be composed of only at least 7 alanine
residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues.
REP may be an amino acid sequence composed of 10 to 200, 10 to 180, 10 to 160, 10
to 140, 10 to 120, 10 to 100, 10 to 80, 10 to 60, or 10 to 40 amino acid residues.
m represents an integer of 2 to 300, and may be an integer of 8 to 300, 10 to 300,
20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to
160, 20 to 140, or 20 to 120. A plurality of (A)
n motifs may have the same amino acid sequence or different amino acid sequences. A
plurality of REPs may have the same amino acid sequence or different amino acid sequences.
[0018] For example, the modified fibroin according to the present embodiment can be obtained
by performing an amino acid sequence modification on a cloned gene sequence of a naturally
derived fibroin, for example, corresponding to substitution, deletion, insertion and/or
addition of one or more amino acid residues. Amino acid residues can be substituted,
deleted, inserted and/or added using methods well known to those skilled in the art
such as partial mutagenesis. Specifically, a method described in the document
Nucleic Acid Res. 10, 6487 (1982),
Methods in Enzymology, 100, 448 (1983) can be used.
[0019] A naturally derived fibroin is a protein including a domain sequence represented
by Formula 1: [(A)
n motif-REP]
m, or Formula 2: [(A)
n motif-REP]
m-(A)
n motif, and specific examples thereof include fibroins produced by insects or spiders.
[0020] Examples of fibroins produced by insects include silk proteins produces by silkworms
such as
Bombyx mori, Kuwako
(Bombyx mandarina), Japanese silk moth
(Antheraea yamamai), tussar silkmoth
(Anteraea pernyi), maple silkworm
(Eriogyna pyretorum), cynthia silkworm
(Pilosamia Cynthia ricini), ailanthus silkmoth
(Samia cynthia), apanese giant silkworm
(Caligura japonica), tasar silkworm
(Antheraea mylitta), and muga silkworm
(Antheraea assama), and hornet silk proteins that are discharged by larvae such as those of the hornet
(Vespa simillima xanthoptera).
[0021] More specific examples of fibroins produced by insects include silkworm fibroin L
chain (GenBank accession number M76430 (nucleotide sequence) and AAA27840.1 (amino
acid sequence)).
[0022] Examples of fibroins produced by spiders include spider silk proteins produced by
spiders belonging to the genus
Araneus such as
Araneus ventricosus, Araneus diadematus, Araneus pentagrammicus, Araneus pentagrammicus and
Araneus nojimai, spiders belonging to the genus
Neoscona such as
Neoscona scylla, Neoscona nautica, Neoscona adianta and
Neoscona scylloides, spiders belonging to the genus
Pronus such as
Pronoides brunneus, spiders belonging to the genus
Cyrtarachne such as
Cyrtarachne bufo and
Cyrtarachne inaequalis, spiders belonging to the genus
Gasteracantha such as
Gasteracantha kuhli and
Thelacantha brevipina, spiders belonging to the genus
Ordgarius such as
Ordgarius hobsoni and
Ordgarius sexspinosus, spiders belonging to the genus
Argiope such as
Argiope amoena, Argiope minuta and
Argiope bruennichi, spiders belonging to the genus
Arachnura such as
Arachnura logio, spiders belonging to the genus
Acusilas such as
Acusilas coccineus, spiders belonging to the genus
Cytophora such as
Cyrtophora ikomosanensis, Cyrtophora exanthematica and
Cyrtophora unicolor, spiders belonging to the genus
Poltys such as
Poltys illepidus, spiders belonging to the genus
Cyclosa such as
Cyclosa octotuberculata, Cyclosa sedeculata, Cyclosa vallata and
Cyclosa atrata, and spiders belonging to the genus
Chorizopes such as
Chorizopes nipponicus, and spider silk proteins produced by spiders belonging to the genus
Tetragnatha such as
Tetragnatha praedonia, Tetragnatha maxillosa, Tetragnatha extensa and
Tetragnatha squamata, spiders belonging to the genus
Leucauge such as
Leucauge celebesiana, Leucauge blanda and
Leucauge subblanda, spiders belonging to the genus
Nephila such as
Nephila clavata and
Nephila pilipes, spiders belonging to the genus
Menosira such as
Menosira ornata, spiders belonging to the genus
Dyschiriognatha such as
Tetragnathidae, spiders belonging to the genus
Latrodectus such as
Latrodectus mactans, Latrodectus hasseltii, Latrodectus geometricus and
Latrodectus tredecimguttat, and spiders belonging to the family
Tetragnathidae such as spiders belonging to the genus
Euprosthenops. Examples of spider silk proteins include dragline silk proteins such as MaSp (MaSpl
and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSpl and MiSp2).
[0023] More specific examples of spider silk proteins produced by spiders include, for example,
fibroin-3(adf-3) [derived from
Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)),
fibroin-4(adf-4) [derived from
Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (nucleotide sequence)),
dragline silk protein spidroin 1 [derived from
Nephila clavipes] (GenBank accession number AAC04504 (amino acid sequence), U37520 (nucleotide sequence)),
major ampullate spidroin 1 [derived from
Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)),
dragline silk protein spidroin 2 [derived from
Nephila clavata] (GenBank accession number AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)),
major ampullate spidroin 1 [derived from
Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)),
and major ampullate spidroin 2
[Euprosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (nucleotide
sequence)), minor ampullate silk protein 1 [
Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk
protein 2 [
Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like
protein [
Nephilengys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence), and the like.
[0024] More specific examples of naturally derived fibroins may further include fibroins
whose sequence information is registered in the NCBI GenBank. For example, within
sequence information registered in the NCBI GenBank, from sequences containing INV
as DIVISION, sequences in which spidroin, ampullate, fibroin, "silk and polypeptide,"
or "silk and protein" are used as keywords in DEFINITION, it can be confirmed that
a character string of a specific product from CDS, and sequences in which a specific
character string is used in TISSUE TYPE from SOURCE are extracted.
[0025] The modified fibroin according to the present embodiment may be a modified silk fibroin
(in which an amino acid sequence of a silk protein produced by silkworms is modified)
or a modified spider silk fibroin (in which an amino acid sequence of a spider silk
protein produced by spiders is modified). Regarding the modified fibroin, a modified
spider silk fibroin is preferable because it has excellent heat retention, hygroscopic
heat generation characteristics and/or flame retardancy.
[0026] Specific examples of modified fibroins include a modified fibroin derived from a
large vesicular transport silk protein produced in the large ampullar gland of a spider
(first modified fibroin), a modified fibroin having a domain sequence with a reduced
content of glycine residues (second modified fibroin), a modified fibroin having a
domain sequence with a reduced content of the (A)
n motif (third modified fibroin), a modified fibroin with a reduced content of glycine
residues and a reduced content of the (A)
n motif (fourth modified fibroin), a modified fibroin having a domain sequence containing
a region having a locally high hydrophobicity index (fifth modified fibroin), and
a modified fibroin having a domain sequence with a reduced content of glutamine residues
(sixth modified fibroin).
[0027] Examples of first modified fibroins include a protein including a domain sequence
represented by Formula 1: [(A)
n motif-REP]
m. In the first modified fibroin, the number of amino acid residues of the (A)
n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20,
still more preferably an integer of 8 to 20, still more preferably an integer of 10
to 20, still more preferably an integer of 4 to 16, particularly preferably an integer
of 8 to 16, and most preferably an integer of 10 to 16. In the first modified fibroin,
in Formula 1, the number of amino acid residues constituting REP is preferably 10
to 200, more preferably 10 to 150, still more preferably 20 to 100, and still more
preferably 20 to 75. In the first modified fibroin, a total number of glycine residues,
serine residues and alanine residues included in the amino acid sequence represented
by Formula 1: [(A)
n motif-REP]
m is preferably 40% or more, more preferably 60% or more, and still more preferably
70% or more with respect to the total number of amino acid residues.
[0028] The first modified fibroin includes a unit of the amino acid sequence represented
by Formula 1: [(A)
n motif-REP]
m and may be a polypeptide in which the C-terminal sequence is an amino acid sequence
shown in any of SEQ ID NOs 1 to 3 or an amino acid sequence having a homology of 90%
or more with an amino acid sequence shown in any of SEQ ID NOs 1 to 3.
[0029] The amino acid sequence shown in SEQ ID NO 1 is the same as the amino acid sequence
composed of amino acids of 50 residues of the C-terminal of the amino acid sequence
of ADF3 (GI: 1263287, NCBI), the amino acid sequence shown in SEQ ID NO 2 is the same
as the amino acid sequence obtained by removing 20 residues from the C-terminal of
the amino acid sequence shown in SEQ ID NO 1, and the amino acid sequence shown in
SEQ ID NO 3 is the same as the amino acid sequence obtained by removing 29 residues
from the C-terminal of the amino acid sequence shown in SEQ ID NO 1.
[0030] More specific examples of first modified fibroins include a modified fibroin including
(1-i) an amino acid sequence shown in SEQ ID NO 4 (recombinant spider silk protein
ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence having a sequence identity of
90% or more with the amino acid sequence shown in SEQ ID NO 4. The sequence identity
is preferably 95% or more.
[0031] The amino acid sequence shown in SEQ ID NO 4 is obtained by performing mutation on
the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO 5) composed
of the start codon, His10 tag and HRV3C protease (Human rhinovirus 3C protease) recognition
site is added to the N-terminal such that the number of 1st to 13th repeat regions
is approximately doubled, and the translation is terminated at the 1,154th amino acid
residue. The amino acid sequence of the C-terminal of the amino acid sequence shown
in SEQ ID NO 4 is the same as the amino acid sequence shown in SEQ ID NO 3.
[0032] The modified fibroin (1-i) may be composed of the amino acid sequence shown in SEQ
ID NO 4.
[0033] As compared with a naturally derived fibroin, in the second modified fibroin, the
domain sequence includes an amino acid sequence with a reduced content of glycine
residues. It can be said that the second modified fibroin has an amino acid sequence
corresponding to a case in which at least one or more glycine residues in REP are
substituted with other amino acid residues, as compared with a naturally derived fibroin.
[0034] As compared with a naturally derived fibroin, in the second modified fibroin, the
domain sequence includes an amino acid sequence corresponding to a case in which,
in at least one motif sequence selected from among GGX and GPGXX (where, G represents
a glycine residue, P represents a proline residue, and X represents an amino acid
residue other than glycine) in REP, one glycine residue in at least one or more motif
sequences is substituted with another amino acid residue.
[0035] In the second modified fibroin, a proportion of the motif sequences in which the
above glycine residue is substituted with another amino acid residue with respect
to all motif sequences may be 10% or more.
[0036] The second modified fibroin includes a domain sequence represented by Formula 1:
[(A)
n motif-REP]
m, and may include an amino acid sequence in which, when the total number of amino
acid residues of amino acid sequences composed of XGX (where, X represents an amino
acid residue other than glycine) contained in all REPs in the sequence obtained by
removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as z, and the total number of amino acid residues
in the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as w, z/w is 30% or more, 40% or more, 50% or more
or 50.9% or more. The number of alanine residues with respect to the total number
of amino acid residues in the (A)
n motif is 83% or more, preferably 86% or more, more preferably 90% or more, still
more preferably 95% or more, and still more preferably 100% (meaning that it is composed
of only alanine residues).
[0037] The second modified fibroin is preferably a modified fibroin in which the proportional
content of amino acid sequences composed of XGX is increased by substituting one glycine
residue of a GGX motif with another amino acid residue. In the second modified fibroin,
the proportional content of amino acid sequences composed of GGX in the domain sequence
is preferably 30% or less, more preferably 20% or less, still more preferably 10%
or less, still more preferably 6% or less, still more preferably 4% or less, and particularly
preferably 2% or less. The proportional content of amino acid sequences composed of
GGX in the domain sequence can be calculated by the same method as the following method
for calculating the proportional content (z/w) of the amino acid sequences composed
of XGX.
[0038] A method for calculating z/w will be described in more detail. First, in a fibroin
(modified fibroin or naturally derived fibroin) including a domain sequence represented
by Formula 1: [(A)
n motif-REP]
m, an amino acid sequence composed of XGX is extracted from all REPs included in the
sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence. The total number of amino acid residues constituting XGX
is z. For example, when 50 amino acid sequences composed of XGX are extracted (no
overlap), z is 50x3=150. In addition, for example, when there is X contained in two
XGXs (X at the center), as in the case of the amino acid sequence composed of XGXGX,
calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid
residues). w is the total number of amino acid residues contained in the sequence
obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence. For example, in the case of the domain sequence shown in
FIG. 1, w is 4+50+4+100+4+10+4+20+4+30=230 (the (A)
n motif positioned closest to the C-terminal side is excluded). Next, z/w (%) can be
calculated by dividing z by w.
[0039] Here, z/w in the naturally derived fibroin will be described. First, as described
above, according to confirmation using the exemplified method in which a fibroin whose
amino acid sequence information is registered in the NCBI GenBank is extracted, 663
types of fibroins (of which 415 types are fibroins produced by spiders) are extracted.
Among all of the extracted fibroins, z/w is calculated from the amino acid sequence
of the naturally derived fibroin including a domain sequence represented by Formula
1: [(A)
n motif-REP]
m and in which the proportional content of amino acid sequence composed of GGX in the
fibroin is 6% or less by the above calculation method. The results are shown in FIG.
2. In FIG. 2, the horizontal axis represents z/w (%), and the vertical axis represents
frequency. As can be clearly understood from FIG. 2, z/w in the naturally derived
fibroin is always less than 50.9% (the highest is 50.86%).
[0040] In the second modified fibroin, z/w is preferably 50.9% or more, more preferably
56.1% or more, still more preferably 58.7% or more, still more preferably 70% or more,
and still more preferably 80% or more. The upper limit of z/w is not particularly
limited, and may be, for example, 95% or less.
[0041] For example, the second modified fibroin can be obtained by performing a modification
on a cloned gene sequence of the naturally derived fibroin such that at least a part
of the nucleotide sequence that encodes a glycine residue is substituted and encodes
another amino acid residue. In this case, regarding the glycine residue to be modified,
one glycine residue in a GGX motif and a GPGXX motif may be selected or substitution
may be performed so that z/w is 50.9% or more. In addition, for example, the second
modified fibroin can be obtained by designing an amino acid sequence that meets the
requirements for the above aspect using the amino acid sequence of a naturally derived
fibroin and chemically synthesizing nucleic acids that encode the designed amino acid
sequence. In any case, in addition to a modification in which a glycine residue in
REP is substituted with another amino acid residue from the amino acid sequence of
a naturally derived fibroin, the amino acid sequence may be additionally modified
such as by substitution, deletion, insertion and/or addition of one or more amino
acid residues.
[0042] The above other amino acid residues are not particularly limited as long as they
are amino acid residues other than glycine residues, and hydrophobic amino acid residues
such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, methionine
(M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W)
residues, and hydrophilic amino acid residue such as glutamine (Q) residues, asparagine
(N) residues, serine (S) residues, lysine (K) residues and glutamic acid (E) residues
are preferable, valine (V) residues, leucine (L) residues, isoleucine (I) residues,
phenylalanine (F) residues and glutamine (Q) residues are more preferable, and glutamine
(Q) residues are still more preferable.
[0043] More specific examples of second modified fibroins include a modified fibroin including
(2-i) an amino acid sequence shown in SEQ ID NO 6 (Met-PRT380), SEQ ID NO 7 (Met-PRT410),
SEQ ID NO 8 (Met-PRT525) or SEQ ID NO 9 (Met-PRT799) or (2-ii) an amino acid sequence
having a sequence identity of 90% or more with an amino acid sequence shown in SEQ
ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
[0044] The modified fibroin (2-i) will be described. In the amino acid sequence shown in
SEQ ID NO 6, all GGXs in REP of the amino acid sequence shown in SEQ ID NO 10 (Met-PRT313)
corresponding to the naturally derived fibroin are substituted with GQX. In the amino
acid sequence shown in SEQ ID NO 7, every two (A)
n motifs from the N-terminal side to the C-terminal side are deleted from the amino
acid sequence shown in SEQ ID NO 6, and additionally, one [(A)
n motif-REP] is inserted before the C-terminal sequence. In the amino acid sequence
shown in SEQ ID NO 8, two alanine residues are inserted into the C-terminal side of
each (A)
n motif of the amino acid sequence shown in SEQ ID NO 7, and additionally, some glutamine
(Q) residues are substituted with serine (S) residues, and some amino acids on the
C-terminal side are deleted so that the molecular weight is almost the same as that
of SEQ ID NO 7. In the amino acid sequence shown in SEQ ID NO 9, a predetermined hinge
sequence and His tag sequence are added to the C-terminal of a sequence obtained by
repeating a region of 20 domain sequences present in the amino acid sequence shown
in SEQ ID NO 7 (where, several amino acid residues on the C-terminal side of the region
are substituted) four times.
[0045] The value of z/w in the amino acid sequence shown in SEQ ID NO 10 (corresponding
to the naturally derived fibroin) is 46.8%. The values of z/w in the amino acid sequence
shown in SEQ ID NO 6, the amino acid sequence shown in SEQ ID NO 7, the amino acid
sequence shown in SEQ ID NO 8, and the amino acid sequence shown in SEQ ID NO 9 are
58.7%, 70.1%, 66.1% and 70.0%, respectively. In addition, the values of x/y at the
Giza ratio (to be described below) of 1:1.8 to 11.3 in the amino acid sequences shown
in SEQ ID NO 10, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9 are 15.0%,
15.0%, 93.4%, 92.7% and 89.8%, respectively.
[0046] The modified fibroin (2-i) may be composed of an amino acid sequence shown in SEQ
ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
[0047] The modified fibroin (2-ii) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 6, SEQ ID NO 7, SEQ
ID NO 8 or SEQ ID NO 9. The modified fibroin (2-ii) is also a protein including a
domain sequence represented by Formula 1: [(A)
n motif-REP]
m. The sequence identity is preferably 95% or more.
[0048] The modified fibroin (2-ii) has a sequence identity of 90% or more with an amino
acid sequence shown in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, and when
the total number of amino acid residues of the amino acid sequence composed of XGX
(where, X represents an amino acid residue other than glycine) contained in REP is
set as z, and the total number of amino acid residues of REP in the above domain sequence
is set as w, z/w is preferably 50.9% or more.
[0049] The second modified fibroin may include a tag sequence at either or both of the N-terminal
and the C-terminal. This makes it possible to isolate, immobilize, detect and visualize
the modified fibroin.
[0050] Regarding the tag sequence, for example, an affinity tag using specific affinity
(binding property, affinity) with other molecules may be exemplified. Specific examples
of affinity tags include a histidine tag (His tag). The His tag is a short peptide
in which about 4 to 10 histidine residues are present and has a property of specifically
binding to metal ions such as nickel, and thus can be used for isolating a modified
fibroin through chelating metal chromatography. Specific examples of tag sequences
include, for example, the amino acid sequence shown in SEQ ID NO 11 (an amino acid
sequence including a His tag sequence and a hinge sequence).
[0051] In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically
binds to glutathione and a maltose binding protein (MBP) that specifically binds to
maltose can be used.
[0052] In addition, an "epitope tag" using an antigen-antibody reaction can be used. When
a peptide (epitope) exhibiting antigenicity is added as a tag sequence, an antibody
against the epitope can be bound. Examples of epitope tags include an HA (a peptide
sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. When
an epitope tag is used, the modified fibroin can be easily purified with high specificity.
[0053] In addition, a tag sequence that can be disconnected with a specific protease can
be used. When a protein adsorbed via the tag sequence is treated with a protease,
it is possible to collect the modified fibroin from which the tag sequence is disconnected.
[0054] More specific examples of modified fibroins containing a tag sequence include a modified
fibroin including (2-iii) an amino acid sequence shown in SEQ ID NO 12 (PRT380), SEQ
ID NO 13 (PRT410), SEQ ID NO 14 (PRT525) or SEQ ID NO 15 (PRT799), or (2-iv) an amino
acid sequence having a sequence identity of 90% or more with an amino acid sequence
shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
[0055] The amino acid sequences shown in SEQ ID NO 16 (PRT313), SEQ ID NO 12, SEQ ID NO
13, SEQ ID NO 14 and SEQ ID NO 15 are obtained by adding the amino acid sequence shown
in SEQ ID NO 11 (including a His tag sequence and a hinge sequence) to the N-terminal
of the amino acid sequences shown in SEQ ID NO 10, SEQ ID NO 6, SEQ ID NO 7, SEQ ID
NO 8 and SEQ ID NO 9, respectively.
[0056] The modified fibroin (2-iii) may be composed of an amino acid sequence shown in SEQ
ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
[0057] The modified fibroin (2-iv) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ
ID NO 14 or SEQ ID NO 15. The modified fibroin (2-iv) is also a protein including
a domain sequence represented by Formula 1: [(A)
n motif-REP]
m. The sequence identity is preferably 95% or more.
[0058] The modified fibroin (2-iv) has a sequence identity of 90% or more with an amino
acid sequence shown in SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15, and
when the total number of amino acid residues of the amino acid sequence composed of
XGX (where, X represents an amino acid residue other than glycine) contained in REP
is set as z, and the total number of amino acid residues of REP in the above domain
sequence is set as w, z/w is preferably 50.9% or more.
[0059] The second modified fibroin may include a secretory signal for releasing the protein
produced in a recombinant protein production system to the outside of the host. The
sequence of the secretory signal can be appropriately set depending on the type of
the host.
[0060] As compared with a naturally derived fibroin, in the third modified fibroin, the
domain sequence includes an amino acid sequence with a reduced content of the (A)
n motif. It can be said that the domain sequence of the third modified fibroin includes
an amino acid sequence corresponding to a case in which at least one or more (A)
n motifs are deleted, as compared with a naturally derived fibroin.
[0061] The third modified fibroin may include an amino acid sequence corresponding to a
case in which 10 to 40% of the (A)
n motif is deleted from the naturally derived fibroin.
[0062] As compared with a naturally derived fibroin, in the third modified fibroin, the
domain sequence may include an amino acid sequence corresponding to a case in which
at least one (A)
n motif for every one to three (A)
n motifs from the N-terminal side to the C-terminal side is deleted.
[0063] As compared with a naturally derived fibroin, in the third modified fibroin, the
domain sequence may include an amino acid sequence corresponding to a case in which,
from the N-terminal side to the C-terminal side, at least deletion of two consecutive
(A)
n motifs and deletion of one (A)
n motif are repeated in that order.
[0064] In the third modified fibroin, the domain sequence may include an amino acid sequence
corresponding to a case in which at least every two (A)
n motifs are deleted from the N-terminal side to the C-terminal side.
[0065] The third modified fibroin includes a domain sequence represented by Formula 1: [(A)
n motif-REP]
m, and may include an amino acid sequence in which, when the numbers of amino acid
residues of REP of adjacent two [(A)
n motif-REP] units are sequentially compared from the N-terminal side to the C-terminal
side, the maximum value of the total value obtained by adding the number of amino
acid residues of the adjacent two [(A)
n motif-REP] units in which a ratio of the number of amino acid residues of the other
REP with respect to 1 set for the number of amino acid residues of one REP with a
smaller number of amino acid residues is 1.8 to 11.3 is set as x, and the total number
of amino acid residues of the domain sequence is set as y, x/y is 20% or more, 30%
or more, 40% or more or 50% or more. The number of alanine residues with respect to
the total number of amino acid residues in the (A)
n motif may be 83% or more, and is preferably 86% or more, more preferably 90% or more,
still more preferably 95% or more, and still more preferably 100% (meaning it is composed
of only alanine residues).
[0066] A method for calculating x/y will be described in more detail with reference to FIG.
1. FIG. 1 shows a domain sequence in which the N-terminal sequence and the C-terminal
sequence are removed from the modified fibroin. The domain sequence includes a sequence
of (A)
n motif-first REP (50 amino acid residues)-(A)
n motif-second REP (100 amino acid residues)-(A)
n motif-third REP (10 amino acid residues)-(A)
n motif-fourth REP (20 amino acid residues)-(A)
n motif-fifth REP (30 amino acid residues)-(A)
n motif from the N-terminal side (left side).
[0067] Adjacent two [(A)
n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal
side so that there is no overlap. In this case, an unselected [(A)
n motif-REP] unit may be present. FIG. 1 shows Pattern 1 (comparison of a first REP
and a second REP, and comparison of a third REP and a fourth REP), Pattern 2 (comparison
of a first REP and a second REP, and comparison of a fourth REP and a fifth REP),
Pattern 3 (comparison of a second REP and a third REP and comparison of a fourth REP
and a fifth REP), and Pattern 4 (comparison of a first REP and a second REP). Here,
there are other selection methods.
[0068] Next, in the patterns, the numbers of amino acid residues of each REP in the selected
adjacent two [(A)
n motif-REP] units are compared. The comparison is performed by calculating a ratio
of the number of amino acid residues of the other REP with respect to 1 set for one
REP with a smaller number of amino acid residues. For example, in the case of comparison
of the first REP (50 amino acid residues) and the second REP (100 amino acid residues),
a ratio of the number of amino acid residues of the second REP with respect to 1 set
for the first REP having a smaller number of amino acid residues is 100/50=2. Similarly,
in the case of comparison of the fourth REP (20 amino acid residues) and the fifth
REP (30 amino acid residues), a ratio of the number of amino acid residues of the
fifth REP with respect to 1 set for the fourth REP with a smaller number of amino
acid residues is 30/20= 1.5.
[0069] In FIG. 1, a set of [(A)
n motif-REP] units in which a ratio of the number of amino acid residues of the other
REP with respect to 1 set for one REP with a smaller number of amino acid residues
is 1.8 to 11.3 is indicated by a solid line. In this specification, this ratio is
called a Giza ratio. A set of [(A)
n motif-REP] units in which a ratio of the number of amino acid residues of the other
REP with respect to 1 set for one REP with a smaller number of amino acid residues
is less than 1.8 or more than 11.3 is indicated by a dotted line.
[0070] In the patterns, the total number of amino acid residues of adjacent two [(A)
n motif-REP] units indicated by a solid line is summed (not only REP but also the number
of amino acid residues in the (A)
n motif). Then, the sum total values are compared, and a total value (a maximum value
of the total value) of the pattern in which the total value is a maximum is set as
x. In the example shown in FIG. 1, the total value of Pattern 1 is a maximum.
[0071] Next, x/y (%) can be calculated by dividing x by the total number y of amino acid
residues of the domain sequence.
[0072] In the third modified fibroin, x/y is preferably 50% or more, more preferably 60%
or more, still more preferably 65% or more, still more preferably 70% or more, still
more preferably 75% or more, and particularly preferably 80% or more. The upper limit
of x/y is not particularly limited, and may be, for example, 100% or less. When the
Giza ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, when the Giza ratio
is 1:1.8 to 3.4, x/y is preferably 77.1% or more, when the Giza ratio is 1:1.9 to
8.4, x/y is preferably 75.9% or more, and when the Giza ratio is 1:1.9 to 4.1, x/y
is preferably 64.2% or more.
[0073] When the third modified fibroin is a modified fibroin in which at least seven of
the plurality of (A)
n motifs present in the domain sequence are composed of only alanine residues, x/y
is preferably 46.4% or more, more preferably 50% or more, still more preferably 55%
or more, still more preferably 60% or more, still more preferably 70% or more, and
particularly preferably 80% or more. The upper limit of x/y is not particularly limited,
and may be 100% or less.
[0074] Here, x/y in the naturally derived fibroin will be described. First, as described
above, according to confirmation using the exemplified method in which a fibroin whose
amino acid sequence information is registered in the NCBI GenBank is extracted, 663
types of fibroins (of which 415 types are fibroins produced by spiders) are extracted.
Among all of the extracted fibroins, x/y is calculated from the amino acid sequence
of the naturally derived fibroin composed of a domain sequence represented by Formula
1: [(A)
n motif-REP]
m according to the above calculation method. The results when the Giza ratio is 1:1.9
to 4.1 are shown in FIG. 3.
[0075] In FIG. 3, the horizontal axis represents x/y (%), and the vertical axis represents
frequency. As can be clearly understood from FIG. 3, x/y in the naturally derived
fibroin is always less than 64.2% (the highest is 64.14%).
[0076] For example, the third modified fibroin can be obtained by deleting one or more sequences
that encode the (A)
n motif from the cloned gene sequence of the naturally derived fibroin so that x/y
is 64.2% or more. In addition, for example, an amino acid sequence corresponding to
a case in which one or more (A)
n motifs are deleted from the amino acid sequence of the naturally derived fibroin
so that x/y is 64.2% or more may be designed, and nucleic acids that encode the designed
amino acid sequence may be chemically synthesized to obtain the third modified fibroin.
In any case, in addition to a modification in which the (A)
n motif is deleted from the amino acid sequence of the naturally derived fibroin, the
amino acid sequence may be additionally modified such as by substitution, deletion,
insertion and/or addition of one or more amino acid residues.
[0077] More specific examples of third modified fibroins include a modified fibroin including
(3-i) an amino acid sequence shown in SEQ ID NO 17 (Met-PRT399), SEQ ID NO 7 (Met-PRT410),
SEQ ID NO 8 (Met-PRT525) or SEQ ID NO 9 (Met-PRT799) or (3-ii) an amino acid sequence
having a sequence identity of 90% or more with an amino acid sequence shown in SEQ
ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
[0078] The modified fibroin (3-i) will be described. In the amino acid sequence shown in
SEQ ID NO 17, every two (A)
n motifs from the N-terminal side to the C-terminal side are deleted from the amino
acid sequence shown in SEQ ID NO 10 (Met-PRT313) corresponding to the naturally derived
fibroin, and additionally, one [(A)
n motif-REP] is inserted before the C-terminal sequence. The amino acid sequences shown
in SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9 are the same as those described in the
second modified fibroin.
[0079] The value of x/y at the Giza ratio of 1:1.8 to 11.3 of the amino acid sequence shown
in SEQ ID NO 10 (corresponding to the naturally derived fibroin) is 15.0%. The values
of x/y in the amino acid sequence shown in SEQ ID NO 17 and the amino acid sequence
shown in SEQ ID NO 7 are always 93.4%. The value of x/y in the amino acid sequence
shown in SEQ ID NO 8 is 92.7%. The value of x/y in the amino acid sequence shown in
SEQ ID NO 9 is 89.8%. The values of z/w in the amino acid sequences shown in SEQ ID
NO 10, SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9 are 46.8%, 56.2%, 70.1%,
66.1% and 70.0%, respectively.
[0080] The modified fibroin (3-i) may be composed of an amino acid sequence shown in SEQ
ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.
[0081] The modified fibroin (3-ii) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 17, SEQ ID NO 7, SEQ
ID NO 8 or SEQ ID NO 9. The modified fibroin (3-ii) is also a protein including a
domain sequence represented by Formula 1: [(A)
n motif-REP]
m. The sequence identity is preferably 95% or more.
[0082] The modified fibroin (3-ii) has a sequence identity of 90% or more with an amino
acid sequence shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9, and
when the numbers of amino acid residues of REP of adjacent two [(A)
n motif-REP] units from the N-terminal side to the C-terminal side are sequentially
compared, the maximum value of the total value obtained by adding the number of amino
acid residues of the adjacent two [(A)
n motif-REP] units in which a ratio of the number of amino acid residues of the other
REP with respect to 1 set for the number of amino acid residues of one REP with a
smaller number of amino acid residues is 1.8 to 11.3 (the Giza ratio is 1:1.8 to 11.3)
is set as x, and the total number of amino acid residues of the domain sequence is
set as y, x/y is preferably 64.2% or more.
[0083] The third modified fibroin may include the above tag sequence at either or both of
the N-terminal and the C-terminal.
[0084] More specific examples of modified fibroins including a tag sequence include a modified
fibroin including (3-iii) an amino acid sequence shown in SEQ ID NO 18 (PRT399), SEQ
ID NO 13 (PRT410), SEQ ID NO 14 (PRT525) or SEQ ID NO 15 (PRT799) or (3-iv) an amino
acid sequence having a sequence identity of 90% or more with an amino acid sequence
shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
[0085] The amino acid sequences shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 and SEQ
ID NO 15 are obtained by adding the amino acid sequence shown in SEQ ID NO 11 (including
a His tag sequence and a hinge sequence) of the N-terminal of the amino acid sequences
shown in SEQ ID NO 17, SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, respectively.
[0086] The modified fibroin (3-iii) may be composed of an amino acid sequence shown in SEQ
ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.
[0087] The modified fibroin (3-iv) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 18, SEQ ID NO 13, SEQ
ID NO 14 or SEQ ID NO 15. The modified fibroin (3-iv) is also a protein including
a domain sequence represented by Formula 1: [(A)
n motif-REP]
m. The sequence identity is preferably 95% or more.
[0088] The modified fibroin (3-iv) has a sequence identity of 90% or more with an amino
acid sequence shown in SEQ ID NO 18, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15, and
when the numbers of amino acid residues of REP of adjacent two [(A)
n motif-REP] units from the N-terminal side to the C-terminal side are sequentially
compared, the maximum value of the total value obtained by adding the number of amino
acid residues of the adjacent two [(A)
n motif-REP] units in which a ratio of the number of amino acid residues of the other
REP with respect to 1 set for the number of amino acid residues of one REP with a
smaller number of amino acid residues is 1.8 to 11.3 is set as x, and the total number
of amino acid residues of the domain sequence is set as y, x/y is preferably 64.2%
or more.
[0089] The third modified fibroin may include a secretory signal for releasing the protein
produced in a recombinant protein production system to the outside of the host. The
sequence of the secretory signal can be appropriately set depending on the type of
the host.
[0090] As compared with a naturally derived fibroin, in the fourth modified fibroin, the
domain sequence includes an amino acid sequence in which the content of the (A)
n motif is reduced and also the content of glycine residues is reduced. As compared
with a naturally derived fibroin, it can be said that the domain sequence of the fourth
modified fibroin includes an amino acid sequence corresponding to a case in which
at least one or more (A)
n motifs are deleted, and additionally, at least one or more glycine residues in REP
are substituted with other amino acid residues. That is, the fourth modified fibroin
is a modified fibroin having characteristics of the above second modified fibroin
and the third modified fibroin in combination. Specific forms and the like are the
same as those described in the second modified fibroin and the third modified fibroin.
[0091] More specific examples of fourth modified fibroins include a modified fibroin including
(4-i) an amino acid sequence shown in SEQ ID NO 7 (Met-PRT410), SEQ ID NO 8 (Met-PRT525),
SEQ ID NO 9 (Met-PRT799), SEQ ID NO 13 (PRT410), SEQ ID NO 14 (PRT525) or SEQ ID NO
15 (PRT799) or (4-ii) an amino acid sequence having a sequence identity of 90% or
more with an amino acid sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ
ID NO 13, SEQ ID NO 14 or SEQ ID NO 15. Specific forms of modified fibroins including
an amino acid sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 13,
SEQ ID NO 14 or SEQ ID NO 15 are as described above.
[0092] As compared with a naturally derived fibroin, in the fifth modified fibroin, the
domain sequence may include an amino acid sequence corresponding to a case in which
one or more amino acid residues in REP are substituted with an amino acid residue
having a large hydrophobicity index and/or one or more amino acid residues having
a large hydrophobicity index are inserted into REP and containing a region having
a locally large hydrophobicity index.
[0093] The region having a locally large hydrophobicity index is preferably composed of
2 to 4 consecutive amino acid residues.
[0094] The above amino acid residue having a large hydrophobicity index is more preferably
an amino acid residue selected from among isoleucine (I), valine (V), leucine (L),
phenylalanine (F), cysteine (C), methionine (M) and alanine (A).
[0095] As compared with a naturally derived fibroin, in the fifth modified fibroin, in addition
to a modification in which one or more amino acid residues in REP are substituted
with an amino acid residue having a large hydrophobicity index and/or one or more
amino acid residues having a large hydrophobicity index are inserted into REP, as
compared with a naturally derived fibroin, the amino acid sequence may be additionally
modified such as by substitution, deletion, insertion and/or addition of one or more
amino acid residues.
[0096] For example, the fifth modified fibroin can be obtained by substituting one or more
hydrophilic amino acid residues (for example, amino acid residues with a negative
hydrophobicity index) in REP from the cloned gene sequence of the naturally derived
fibroin with hydrophobic amino acid residues (for example, amino acid residues with
a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid
residues into REP. In addition, for example, the fifth modified fibroin can be obtained
by designing an amino acid sequence in which one or more hydrophilic amino acid residues
in REP from the amino acid sequence of the naturally derived fibroin are substituted
with hydrophobic amino acid residues and/or one or more hydrophobic amino acid residues
are inserted into REP, and chemically synthesizing nucleic acids that encode the designed
amino acid sequence. In any case, in addition to a modification in which one or more
hydrophilic amino acid residues in REP are substituted with hydrophobic amino acid
residues from the amino acid sequence of the naturally derived fibroin and/or one
or more hydrophobic amino acid residues are inserted into REP, the amino acid sequence
may be additionally modified such as by substitution, deletion, insertion and/or addition
of one or more amino acid residues.
[0097] The fifth modified fibroin includes a domain sequence represented by Formula 1: [(A)
n motif-REP]
m, and may include an amino acid sequence in which, when the total number of amino
acid residues included in a region in which the average value of the hydrophobicity
index of 4 consecutive amino acid residues is 2.6 or more in all REPs included in
the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as p, and the total number of amino acid residues
included in the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as q, p/q is 6.2% or more.
[0098] Regarding the hydrophobicity index of the amino acid residue, a known index (Hydropathy
index:
Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character
of a protein," J. Mol. Biol., 157, pp. 105-132) may be used. Specifically, the hydrophobicity index (hydropathy index, hereinafter
referred to as "HI") of each amino acid is as shown in the following Table 1.
[Table 1]
Amino acid |
HI |
Amino acid |
HI |
Isoleucine (Ile) |
4.5 |
Tryptophan (Trp) |
-0.9 |
Valine (Val) |
4.2 |
Tyrosine (Tyr) |
-1.3 |
Leucine (Leu) |
3.8 |
Proline (Pro) |
-1.6 |
Phenylalanine (Phe) |
2.8 |
Histidine (His) |
-3.2 |
Cysteine (Cys) |
2.5 |
Asparagine (Asn) |
-3.5 |
Methionine (Met) |
1.9 |
Aspartic acid (Asp) |
-3.5 |
Alanine (Ala) |
1.8 |
Glutamine (Gln) |
-3.5 |
Glycine (Gly) |
-0.4 |
Glutamic acid (Glu) |
-3.5 |
Threonine (Thr) |
-0.7 |
Lysine (Lys) |
-3.9 |
Serine (Ser) |
-0.8 |
Arginine (Arg) |
-4.5 |
[0099] A method for calculating p/q will be described in more detail. For calculation, the
sequence (hereinafter referred to as a "sequence A") obtained by removing a sequence
from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence represented by Formula 1: [(A)
n motif-REP]
m is used. First, in all REPs included in the sequence A, the average value of the
hydrophobicity index of 4 consecutive amino acid residues is calculated. The average
value of the hydrophobicity index is obtained by dividing the sum of HI of amino acid
residues included in 4 consecutive amino acid residues by 4 (the number of amino acid
residues). The average value of the hydrophobicity index is obtained for all 4 consecutive
amino acid residues (each amino acid residue is used for calculating the average value
1 to 4 times). Next, a region in which the average value of the hydrophobicity index
of 4 consecutive amino acid residues is 2.6 or more is specified. Even if some amino
acid residues correspond to a plurality of "4 consecutive amino acid residues in which
the average value of the hydrophobicity index is 2.6 or more," they are included in
the region as one amino acid residue. Thus, the total number of amino acid residues
included in the region is p. In addition, the total number of amino acid residues
included in the sequence A is q.
[0100] For example, when 20 "4 consecutive amino acid residues in which the average value
of the hydrophobicity index is 2.6 or more" are extracted (no overlap), 20 of 4 consecutive
amino acid residues (no overlap) are included in the region in which the average value
of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more, and
p is 20x4=80. In addition, for example, when two "4 consecutive amino acid residues
in which the average value of the hydrophobicity index is 2.6 or more" overlap by
one amino acid residue, 7 amino acid residues are included in the region in which
the average value of the hydrophobicity index of 4 consecutive amino acid residues
is 2.6 or more (p=2×4-1=7, "-1" is deduction for overlap). For example, in the case
of the domain sequence shown in FIG. 4, since there are seven "4 consecutive amino
acid residues in which the average value of the hydrophobicity index is 2.6 or more"
without overlap, p is 7x4=28. In addition, for example, in the case of the domain
sequence shown in FIG. 4, q is 4+50+4+40+4+10+4+20+4+30=170 ((A)
n motif present at the end of the C-terminal side is not included). Next, p/q (%) can
be calculated by dividing p by q. In FIG. 4, 28/170=16.47%.
[0101] In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7%
or more, still more preferably 10% or more, still more preferably 20% or more, and
still more preferably 30% or more. The upper limit of p/q is not particularly limited,
and may be, for example, 45% or less.
[0102] For example, the fifth modified fibroin can be obtained by performing a modification
on a cloned amino acid sequence of the naturally derived fibroin such that one or
more hydrophilic amino acid residues (for example, amino acid residues having a negative
hydrophobicity index) in REP are substituted with hydrophobic amino acid residues
(for example, amino acid residues having a positive hydrophobicity index) and/or one
or more hydrophobic amino acid residues are inserted into REP so that the above p/q
condition is satisfied, and thus a modification to an amino acid sequence including
a region having a locally large hydrophobicity index is performed. In addition, for
example, the fifth modified fibroin can be obtained by designing an amino acid sequence
that satisfies the above p/q condition from the amino acid sequence of the naturally
derived fibroin, and chemically synthesizing nucleic acids that encode the designed
amino acid sequence. In any case, as compared with a naturally derived fibroin, in
addition to a modification in which one or more amino acid residues in REP are substituted
with an amino acid residue having a large hydrophobicity index and/or one or more
amino acid residues having a large hydrophobicity index are inserted into REP, a modification
such as substitution, deletion, insertion and/or addition of one or more amino acid
residues are may be additionally performed.
[0103] The amino acid residue having a large hydrophobicity index is not particularly limited,
and is preferably isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine
(C), methionine (M) or alanine (A), and more preferably valine (V), leucine (L) or
isoleucine (I).
[0104] More specific examples of fifth modified fibroins include a modified fibroin including
(5-i) an amino acid sequence shown in SEQ ID NO 19 (Met-PRT720), SEQ ID NO 20 (Met-PRT665)
or SEQ ID NO 21 (Met-PRT666) or (5-ii) an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or
SEQ ID NO 21.
[0105] The modified fibroin (5-i) will be described. The amino acid sequence shown in SEQ
ID NO 19 is obtained by, with respect to the amino acid sequence shown in SEQ ID NO
7 (Met-PRT410), removing a domain sequence at the terminal of the C-terminal side,
inserting two amino acid sequences (VLI) each composed of three amino acid residues
into every one REP, additionally, substituting some glutamine (Q) residues with serine
(S) residues, and deleting a part of the amino acid on the C-terminal side. The amino
acid sequence shown in SEQ ID NO 20 is obtained by inserting one amino acid sequence
(VLI) composed of three amino acid residues into every one REP of the amino acid sequence
shown in SEQ ID NO 8 (Met-PRT525). The amino acid sequence shown in SEQ ID NO 21 is
obtained by inserting two amino acid sequences (VLI) each composed of three amino
acid residues into every REP of the amino acid sequence shown in SEQ ID NO 8.
[0106] The modified fibroin (5-i) may be composed of an amino acid sequence shown in SEQ
ID NO 19, SEQ ID NO 20 or SEQ ID NO 21.
[0107] The modified fibroin (5-ii) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or
SEQ ID NO 21. The modified fibroin (5-ii) is also a protein including a domain sequence
represented by Formula 1: [(A)
n motif-REP]
m. The sequence identity is preferably 95% or more.
[0108] The modified fibroin (5-ii) has a sequence identity of 90% or more with an amino
acid sequence shown in SEQ ID NO 19, SEQ ID NO 20 or SEQ ID NO 21, and in all REPs
included in the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, when the total number of amino acid residues included in
a region in which the average value of the hydrophobicity index of 4 consecutive amino
acid residues is 2.6 or more is set as p, and the total number of amino acid residues
included in the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as q, p/q is preferably 6.2% or more.
[0109] The fifth modified fibroin may include a tag sequence at either or both of the N-terminal
and the C-terminal.
[0110] More specific examples of modified fibroins including a tag sequence include a modified
fibroin including (5-iii) an amino acid sequence shown in SEQ ID NO 22 (PRT720), SEQ
ID NO 23 (PRT665) or SEQ ID NO 24 (PRT666) or (5-iv) an amino acid sequence having
a sequence identity of 90% or more with an amino acid sequence shown in SEQ ID NO
22, SEQ ID NO 23 or SEQ ID NO 24.
[0111] The amino acid sequences shown in SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24 are
obtained by adding the amino acid sequence shown in SEQ ID NO 11 (including a His
tag sequence and a hinge sequence) of the N-terminal of the amino acid sequences shown
in SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21, respectively.
[0112] The modified fibroin (5-iii) may be composed of an amino acid sequence shown in SEQ
ID NO 22, SEQ ID NO 23 or SEQ ID NO 24.
[0113] The modified fibroin (5-iv) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 22, SEQ ID NO 23 or
SEQ ID NO 24. The modified fibroin (5-iv) is also a protein including a domain sequence
represented by Formula 1: [(A)
n motif-REP]
m. The sequence identity is preferably 95% or more.
[0114] The modified fibroin (5-iv) has a sequence identity of a 90% or more with an amino
acid sequence shown in SEQ ID NO 22, SEQ ID NO 23 or SEQ ID NO 24, and in all REPs
included in the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, when the total number of amino acid residues included in
a region in which the average value of the hydrophobicity index of 4 consecutive amino
acid residues is 2.6 or more is set as p, and the total number of amino acid residues
included in the sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is set as q, p/q is preferably 6.2% or more.
[0115] The fifth modified fibroin may include a secretory signal for releasing the protein
produced in a recombinant protein production system to the outside of the host. The
sequence of the secretory signal can be appropriately set depending on the type of
the host.
[0116] The sixth modified fibroin includes an amino acid sequence in which the content of
glutamine residues is reduced as compared with a naturally derived fibroin.
[0117] The sixth modified fibroin preferably includes at least one motif selected from among
a GGX motif and a GPGXX motif in the amino acid sequence of REP.
[0118] When the sixth modified fibroin includes a GPGXX motif in REP, the content of the
GPGXX motif is generally 1% or more, and may be 5% or more and is preferably 10% or
more. The upper limit of the content of the GPGXX motif is not particularly limited,
and may be 50% or less or 30% or less.
[0119] In this specification, the "content of the GPGXX motif" is a value calculated by
the following method.
[0120] In a fibroin (a modified fibroin or a naturally derived fibroin) including a domain
sequence represented by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif, in all REPs included in the sequence obtained by removing a sequence from
the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, when a number obtained by tripling the total number of GPGXX
motifs included in the region (that is, corresponding to the total number of G and
P in the GPGXX motif) is set as s, and the total number of amino acid residues of
all REPs in which the sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
is removed from the domain sequence, and the (A)
n motif is additionally removed is set as t, the content of the GPGXX motif is calculated
as s/t.
[0121] In calculation of the content of the GPGXX motif, the "sequence obtained by removing
a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" is set as a target because a sequence having a low correlation
with the characteristic sequence of the fibroin is included in the "sequence from
the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence"
(sequence corresponding to REP), and when m is small (that is, when the domain sequence
is short), since this affects the calculation result of the content of the GPGXX motif,
the setting is performed for eliminating this effect. Here, when the "GPGXX motif"
is positioned at the C-terminal of REP, it is treated as a "GPGXX motif" even if "XX"
is, for example, "AA."
[0122] FIG. 5 is a schematic view showing a domain sequence of a modified fibroin. A method
for calculating a content of the GPGXX motif will be described in detail with reference
to FIG. 5. First, in the domain sequence of the modified fibroin shown in FIG. 5 ("[(A)
n motif-REP]
m-(A)
n motif" type), since all REPs are included in the "sequence obtained by removing a
sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" (in FIG. 5, a sequence shown in a "region A"), the number
of GPGXX motifs for calculating s is 7, and s is 7×3=21. Similarly, since all REPs
are included in the "sequence obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" (in FIG. 5, the sequence shown in the "region A"), the total
number t of amino acid residues of all REPs in which the (A)
n motif is additionally removed from the sequence is 50+40+10+20+30=150. Then, s/t
(%) can be calculated by dividing s by t, and is 21/150=14.0% in the case of the modified
fibroin in FIG. 5.
[0123] The glutamine residue content of the sixth modified fibroin is preferably 9% or less,
more preferably 7% or less, still more preferably 4% or less, and particularly preferably
0%.
[0124] In this specification, the "content of glutamine residues" is a value that is calculated
by the following method.
[0125] In a fibroin (a modified fibroin or a naturally derived fibroin) including a domain
sequence represented by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif, in all REPs included in the sequence (the sequence corresponding to the "region
A" in FIG. 5) obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, the total number of glutamine residues included in this
region is set as u, and the total number of amino acid residues of all REPs in which
the sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
is removed from the domain sequence, and the (A)
n motif is additionally removed is set as t, the content of glutamine residues is calculated
as u/t. In calculation of the content of glutamine residues, the reason why the "sequence
obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" is set as a target is the same as above.
[0126] As compared with a naturally derived fibroin, in the sixth modified fibroin, the
domain sequence may include an amino acid sequence corresponding to a case in which
one or more glutamine residues in REP are deleted or substituted with other amino
acid residues.
[0127] The "other amino acid residue" may be any amino acid residue other than the glutamine
residues, but is preferably an amino acid residue having a larger hydrophobicity index
than the glutamine residue. The hydrophobicity index of the amino acid residues is
as shown in Table 1.
[0128] As shown in Table 1, regarding the amino acid residue having a larger hydrophobicity
index than the glutamine residue, amino acid residues selected from among isoleucine
(I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M)alanine
(A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline
(P) and histidine (H) may be exemplified. Among these, an amino acid residue selected
from among isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C),
methionine (M) and alanine (A) is more preferable, and an amino acid residue selected
from among isoleucine (I), valine (V), leucine (L) and phenylalanine (F) is still
more preferable.
[0129] In the sixth modified fibroin, the hydrophobicity of REP is preferably -0.8 or more,
more preferably -0.7 or more, still more preferably 0 or more, still more preferably
0.3 or more, and particularly preferably 0.4 or more. The upper limit of the hydrophobicity
of REP is not particularly limited, and may be 1.0 or less or 0.7 or less.
[0130] In this specification, the "hydrophobicity of REP" is a value calculated by the following
method.
[0131] In a fibroin (a modified fibroin or a naturally derived fibroin) including a domain
sequence represented by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif, in all REPs included in the sequence (the sequence corresponding to the "region
A" in FIG. 5) obtained by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, when the sum of the hydrophobicity indexes of the amino
acid residues in this region is set as v, and the total number of amino acid residues
of all REPs in which the sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
is removed from the domain sequence, and the (A)
n motif is additionally removed is set as t, the hydrophobicity of REP is calculated
as v/t. In calculation of hydrophobicity of REP, the reason why the "sequence obtained
by removing a sequence from the (A)
n motif positioned closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" is set as a target is the same as above.
[0132] As compared with a naturally derived fibroin, in the sixth modified fibroin, in the
domain sequence, in addition to a modification in which one or more glutamine residues
in REP are deleted and/or one or more glutamine residues in REP are substituted with
other amino acid residues, the amino acid sequence may be additionally modified such
as by substitution, deletion, insertion and/or addition of one or more amino acid
residues.
[0133] For example, the sixth modified fibroin can be obtained by deleting one or more glutamine
residues in REP from the cloned gene sequence of the naturally derived fibroin and/or
substituting one or more glutamine residues in REP with other amino acid residues.
In addition, for example, the sixth modified fibroin can be obtained by designing
an amino acid sequence corresponding to a case in which one or more glutamine residues
in REP are deleted from the amino acid sequence of the naturally derived fibroin and/or
one or more glutamine residues in REP are substituted with other amino acid residues
and chemically synthesizing nucleic acids that encode the designed amino acid sequence.
[0134] More specific examples of sixth modified fibroins include (6-i) a modified fibroin
including an amino acid sequence shown in SEQ ID NO 25 (Met-PRT888), SEQ ID NO 26
(Met-PRT965), SEQ ID NO 27 (Met-PRT889), SEQ ID NO 28 (Met-PRT916), SEQ ID NO 29 (Met-PRT918),
SEQ ID NO 30 (Met-PRT699), SEQ ID NO 31 (Met-PRT698) or SEQ ID NO 32 (Met-PRT966)
or (6-ii) a modified fibroin including an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 25, SEQ ID NO 26, SEQ
ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 or SEQ ID NO 32.
[0135] The modified fibroin (6-i) will be described. The amino acid sequence shown in SEQ
ID NO 25 is obtained by substituting all QQs in the amino acid sequence shown in SEQ
ID NO 7 (Met-PRT410) with VL. The amino acid sequence shown in SEQ ID NO 26 is obtained
by substituting all QQs in the amino acid sequence shown in SEQ ID NO 7 with TS and
substituting the remaining Q with A. The amino acid sequence shown in SEQ ID NO 27
is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO
7 with VL and substituting the remaining Q with I. The amino acid sequence shown in
SEQ ID NO 28 is obtained by substituting all QQs in the amino acid sequence shown
in SEQ ID NO 7 with VI and substituting the remaining Q with L. The amino acid sequence
shown in SEQ ID NO 29 is obtained by substituting all QQs in the amino acid sequence
shown in SEQ ID NO 7 with VF and substituting the remaining Q with I.
[0136] The amino acid sequence shown in SEQ ID NO 30 is obtained by substituting all QQs
in the amino acid sequence shown in SEQ ID NO 8 (Met-PRT468) with VL. The amino acid
sequence shown in SEQ ID NO 31 is obtained by substituting all QQs in the amino acid
sequence shown in SEQ ID NO 8 with VL and substituting the remaining Q with I.
[0137] The amino acid sequence shown in SEQ ID NO 32 is obtained by substituting all QQs
in a sequence in which a region including 20 domain sequences in the amino acid sequence
shown in SEQ ID NO 7 (Met-PRT410) is repeated twice with VF and substituting the remaining
Q with I.
[0138] All of the amino acid sequences shown in SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27,
SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32 have a glutamine
residue content of 9% or less (Table 2).
[Table 2]
Modified fibroin |
Glutamine residue content |
GPGXX motif content |
Hydrophobicity of REP |
Met-PRT410 (SEQ ID NO 7) |
17.7% |
27.9% |
-1.52 |
Met-PRT888 (SEQ ID NO 25) |
6.3% |
27.9% |
-0.07 |
Met-PRT965 (SEQ ID NO 26) |
0.0% |
27.9% |
-0.65 |
Met-PRT889 (SEQ ID NO 27) |
0.0% |
27.9% |
0.35 |
Met-PRT916 (SEQ ID NO 28) |
0.0% |
27.9% |
0.47 |
Met-PRT918 (SEQ ID NO 29) |
0.0% |
27.9% |
0.45 |
Met-PRT699 (SEQ ID NO 30) |
3.6% |
26.4% |
-0.78 |
Met-PRT698 (SEQ ID NO 31) |
0.0% |
26.4% |
-0.03 |
Met-PRT966 (SEQ ID NO 32) |
0.0% |
28.0% |
0.35 |
[0139] The modified fibroin (6-i) may be composed of an amino acid sequence shown in SEQ
ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ
ID NO 31 or SEQ ID NO 32.
[0140] The modified fibroin (6-ii) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 25, SEQ ID NO 26, SEQ
ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 or SEQ ID NO 32.
The modified fibroin (6-ii) is also a protein including a domain sequence represented
by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif. The sequence identity is preferably 95% or more.
[0141] The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less.
In addition, the modified fibroin (6-ii) preferably has a GPGXX motif content of 10%
or more.
[0142] The sixth modified fibroin may include a tag sequence at either or both of the N-terminal
and the C-terminal. This makes it possible to isolate, immobilize, detect and visualize
the modified fibroin.
[0143] More specific examples of modified fibroins including a tag sequence include (6-iii)
a modified fibroin including an amino acid sequence shown in SEQ ID NO 33 (PRT888),
SEQ ID NO 34 (PRT965), SEQ ID NO 35 (PRT889), SEQ ID NO 36 (PRT916), SEQ ID NO 37
(PRT918), SEQ ID NO 38 (PRT699), SEQ ID NO 39 (PRT698) or SEQ ID NO 40 (PRT966) or
(6-iv) a modified fibroin including an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 33, SEQ ID NO 34, SEQ
ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40.
[0144] The amino acid sequences shown in SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID
NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 and SEQ ID NO 40 are obtained by adding
the amino acid sequence shown in SEQ ID NO 11 (including a His tag sequence and a
hinge sequence) to the N-terminal of the amino acid sequences shown in SEQ ID NO 25,
SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31
and SEQ ID NO 32, respectively. Since only the tag sequence is added to the N-terminal,
the glutamine residue content does not change, and the amino acid sequences shown
in SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO
38, SEQ ID NO 39 and SEQ ID NO 40 all have a glutamine residue content of 9% or less
(Table 3).
[Table 3]
Modified fibroin |
Glutamine residue content |
GPGXX motif content |
Hydrophobicity of REP |
PRT888 (SEQ ID NO 33) |
6.3% |
27.9% |
-0.07 |
PRT965 (SEQ ID NO 34) |
0.0% |
27.9% |
-0.65 |
PRT889 (SEQ ID NO 35) |
0.0% |
27.9% |
0.35 |
PRT916 (SEQ ID NO 36) |
0.0% |
27.9% |
0.47 |
PRT918 (SEQ ID NO 37) |
0.0% |
27.9% |
0.45 |
PRT699 (SEQ ID NO 38) |
3.6% |
26.4% |
-0.78 |
PRT698 (SEQ ID NO 39) |
0.0% |
26.4% |
-0.03 |
PRT966 (SEQ ID NO 40) |
0.0% |
28.0% |
0.35 |
[0145] The modified fibroin (6-iii) may be composed of an amino acid sequence shown in SEQ
ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ
ID NO 39 or SEQ ID NO 40.
[0146] The modified fibroin (6-iv) includes an amino acid sequence having a sequence identity
of 90% or more with an amino acid sequence shown in SEQ ID NO 33, SEQ ID NO 34, SEQ
ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40.
The modified fibroin (6-iv) is also a protein including a domain sequence represented
by Formula 1: [(A)
n motif-REP]
m or Formula 2: [(A)
n motif-REP]
m-(A)
n motif. The sequence identity is preferably 95% or more.
[0147] The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less.
In addition, the modified fibroin (6-iv) preferably has a GPGXX motif content of 10%
or more.
[0148] The sixth modified fibroin may include a secretory signal for releasing the protein
produced in a recombinant protein production system to the outside of the host. The
sequence of the secretory signal can be appropriately set depending on the type of
the host.
[0149] The modified fibroin may be a modified fibroin having at least two or more characteristics
among characteristics of the first modified fibroin, the second modified fibroin,
the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin,
and the sixth modified fibroin in combination.
[0150] The limit oxygen index (LOI) value of the modified fibroin according to the present
embodiment may be 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or
more, 29 or more, or 30 or more. In this specification, the LOI value is a value measured
according to a test method for powder granules or a synthetic resin having a low melting
point,
Fire Department Dangerous Goods Regulation Division Fire Department 50, May 31, 1995.
[0151] The maximum hygroscopic heat generation of the modified fibroin according to the
present embodiment determined according to the following Formula A may be greater
than 0.025°C/g.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0007)
[0152] Here, in Formula A, the low humidity environment means an environment of a temperature
of 20°C and a relative humidity of 40%, and the high humidity environment means an
environment of a temperature of 20°C and a relative humidity of 90%
[0153] The maximum hygroscopic heat generation of the modified fibroin according to the
present embodiment may be 0.026°C/g or more, 0.027°C/g or more, 0.028°C/g or more,
0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, or 0.040°C/g or more. The
upper limit of the maximum hygroscopic heat generation is not particularly limited,
and is generally 0.060°C/g or less.
[0154] The heat retention index of the modified fibroin according to the present embodiment
determined according to the following Formula B may be 0.20 or more.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0008)
[0155] Here, in this specification, the heat retention rate means a heat retention rate
measured by a dry contact method using Thermo labo-II tester (under a wind of 30 cm/sec),
and is a value measured by a method described in the section of examples to be descried
below.
[0156] The heat retention index of the modified fibroin according to the present embodiment
may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or more, or 0.32
or more. The upper limit of the heat retention index is not particularly limited,
and may be, for example, 0.60 or less or 0.40 or less.
<Method for manufacturing modified fibroin>
[0157] For example, the modified fibroin according to the present embodiment can be produced
by expressing the nucleic acid by a host transformed with an expression vector including
a nucleic acid sequence that encodes the modified fibroin and one or more regulatory
sequences that are operably linked to the nucleic acid sequence.
[0158] A method for manufacturing a nucleic acid that encodes the modified fibroin is not
particularly limited. For example, the nucleic acid can be manufactured by a method
in which a gene that encodes a natural fibroin is used, amplification and cloning
are performed using a polymerase chain reaction (PCR), and modification is performed
by a genetic engineering technique or a chemically synthesizing method. A method for
chemically synthesizing nucleic acids is not particularly limited, and for example,
the gene can be chemically synthesized by a method for linking an oligonucleotide
automatically synthesized using AKTA oligopilot plus 10/100 (commercially available
from GE Healthcare Japan) through PCR based on amino acid sequence information of
fibroins available in the NCBI web database and the like. In this case, in order to
facilitate purification and/or confirmation of the modified fibroin, a nucleic acid
that encodes a modified fibroin composed of an amino acid sequence obtained by adding
an amino acid sequence including a start codon and a His10 tag to the N-terminal of
the amino acid sequence may be synthesized.
[0159] The regulatory sequence is a sequence (for example, a promoter, an enhancer, a ribosome
binding sequence, a transcription termination sequence, etc.) that controls expression
of the modified fibroin in the host, and can be appropriately selected depending on
the type of the host. Regarding the promoter, an inducible promoter that functions
in host cells and can express and induce the modified fibroin may be used. The inducible
promoter is a promoter that can control transcription according to the presence of
an inducing substance (expression inducer), the absence of repressor molecules, or
physical factors such as an increase or decrease in the temperature, the osmotic pressure
or the pH value.
[0160] The type of the expression vector may be a plasmid vector, a virus vector, a cosmid
vector, a fosmid vector, an artificial chromosome vector or the like, and can be appropriately
selected depending on the type of the host. Regarding the expression vector, a vector
which can autonomously replicate in host cells or can be integrated into host chromosomes
and contains a promoter at a position at which a nucleic acid that encodes the modified
fibroin can be transcripted is suitably used.
[0161] Regarding the host, any of prokaryotes and eukaryotes such as yeast, filamentous
fungus, insect cells, animal cells and plant cells can be suitably used.
[0162] Preferable examples of prokaryotic hosts include bacteria belonging to the genus
Escherichia, the genus
Brevibacillus, the genus
Serratia, the genus
Bacillus, the genus
Mycobacterium, the genus
Corynebacterium, the genus
Corynebacterium and the genus
Pseudomonas. Examples of microorganisms belonging to the genus
Escherichia include
Escherichia coli. Examples of microorganisms belonging to the genus
Brevibacillus include
Brevibacillus Agri. Examples of microorganisms belonging to the genus
Serratia include
Serratia liquefaciens. Examples of microorganism belonging to the genus
Bacillus include
Bacillus subtilis. Examples of microorganisms belonging to the genus
Mycobacterium include
Microbacterium ammoniaphilum. Examples of microorganisms belonging to the genus
Corynebacterium include
Brevibacterium divaricatum. Examples of microorganisms belonging to the genus
Corynebacterium include
corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus
Pseudomonas include
Pseudomonas putida.
[0163] When prokaryotes are used as a host, examples of a vector for introducing a nucleic
acid that encodes a modified fibroin include pBTrp2 (commercially available from Boehringer
Ingelheim GmbH), pGEX (commercially available from Pharmacia), pUC18, pBluescriptII,
pSupex, pET22b, pCold, pUB110, and pNCO2 (Japanese Unexamined Patent Publication No.
2002-238569).
[0164] Examples of eukaryotic hosts include yeast and filamentous fungus (mold, etc.). Examples
of yeasts include yeasts belonging to the genus
Saccharomyces, the genus
Pichia, and the genus
Schizosaccharomyces. Examples of filamentous fungi include filamentous fungi belonging to the genus
Aspergillus, the genus
Penicillium, and the genus
Trichoderma.
[0165] When eukaryotes are used as a host, regarding a vector that introduces a nucleic
acid that encodes the modified fibroin, for example, YEP13 (ATCC37115) and YEp24 (ATCC37051)
may be exemplified. Regarding a method for introducing an expression vector into the
host cell, any method can be used as long as it is a method for introducing DNA into
the host cell. For example, a method using calcium ions [
Proc. Natl. Acad. Sci. USA, 69, 2110(1972)], an electroporation method, a spheroplast method, a protoplast method, a lithium
acetate method, a competent method, and the like may be exemplified.
[0166] Regarding a method for expressing a nucleic acid by a host transformed with an expression
vector, in addition to direct expression, secretory production, fusion protein expression,
or the like can be performed according to a method described in Molecular Cloning
2
nd edition or the like.
[0167] For example, the modified fibroin can be manufactured by culturing a host transformed
with an expression vector in a culture medium, generating and storing the modified
fibroin in the culture medium, and collecting the modified fibroin from the culture
medium. A method for culturing a host in the culture medium can be performed according
to a method that is generally used in culturing of the host.
[0168] When the host is prokaryotes such as
E. coli or eukaryotes such as yeast, regarding the culture medium, any of a natural culture
medium and a synthetic culture medium may be used as long as the culture medium contains
a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated
by the host and in which the host is efficiently cultured.
[0169] Regarding the carbon source, any source that can be assimilated by the transformed
microorganism may be used, and for example, glucose, fructose, sucrose, and molasses
containing them, carbohydrates such as starch and starch hydrolyzate, organic acids
such as acetic acid and propionic acid, and alcohols such as ethanol and propanol
can be used. Regarding the nitrogen source, for example, inorganic acids such as ammonia,
ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or ammonium
salts of organic acids, other nitrogen-containing compounds, and peptone, meat extracts,
yeast extracts, corn steep liquors, casein hydrolyzates, soybean cakes and soybean
cake hydrolyzates, various fermented bacteria and their digested products can be used.
Regarding inorganic salts, for example, potassium phosphate, dibasic potassium phosphate,
magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese
sulfate, copper sulfate and calcium carbonate can be used.
[0170] Prokaryotes such as
E. coli or eukaryotes such as yeast can be cultured, for example, under aerobic conditions
such as shaking culture or deep aeration stirring culture. The culture temperature
is, for example, 15 to 40°C. The culture time is generally 16 hours to 7 days. The
pH of a culture medium during culturing is preferably maintained at 3.0 to 9.0. The
pH of the culture medium can be adjusted using an inorganic acid, an organic acid,
an alkaline solution, urea, calcium carbonate, ammonia, and the like.
[0171] In addition, during culturing, as necessary, antibiotics such as ampicillin and tetracycline
may be added to the culture medium. When microorganisms transformed with an expression
vector using an inducible promoter as a promoter are cultured, as necessary, an inducer
may be added to the culture medium. For example, when microorganisms transformed with
an expression vector using a lac promoter are cultured, isopropyl-β-D-thiogalactopyranoside
or the like may be added to the culture medium, and when microorganisms transformed
with an expression vector using a trp promoter are cultured, indole acrylic acid or
the like may be added to the culture medium.
[0172] The expressed modified fibroin can be isolated and purified by a commonly used method.
For example, when the modified fibroin is expressed in a dissolved state in cells,
after culturing, host cells are collected by centrifugation, and suspended in an aqueous
buffer solution, and the host cells are then crushed with an ultrasonic crusher, a
French press, a Manton-Gaulin homogenizer, Dyno Mill or the like, and thereby a cell-free
extract liquid is obtained. Methods commonly used for isolating and purifying the
modified fibroin from a supernatant obtained by centrifuging the cell-free extract
liquid, that is, methods such as a solvent extraction method, a salting-out method
using ammonium sulfate or the like, a desalting method, a precipitation method using
an organic solvent, an anion exchange chromatography method using a resin such as
diethylaminoethyl (DEAE)-sepharose and DIAION HPA-75 (commercially available from
Mitsubishi Chemical Corporation), a cation exchange chromatography method using a
resin such as S-Sepharose FF (commercially available from Pharmacia), a hydrophobic
chromatography method using a resin such as butyl sepharose and phenyl sepharose,
a gel filtration method using a molecular sieve, an affinity chromatography method,
a chromatofocusing method, and an electrophoresis method such as an isoelectric point
electrophoresis method may be used alone or in combination, and thereby a purified
preparation can be obtained.
[0173] In addition, when the modified fibroin is expressed by forming an insoluble component
in cells, host cells are collected in the same manner, and then crushed and centrifuged,
and thus insoluble components of the modified fibroin are collected as a precipitate
fraction. The collected insoluble components of the modified fibroin can be solubilized
with a protein denaturation agent. After the operation, a purified preparation of
the modified fibroin can be obtained by the same isolation and purification method
as described above. When the modified fibroin is secreted extracellularly, the modified
fibroin can be collected from a culture supernatant. That is, the culture product
is treated with a technique such as centrifugation to obtain a culture supernatant,
and a purified preparation can be obtained from the culture supernatant using the
same isolation and purification method as described above.
<Method for manufacturing modified fibroin fiber (filament)>
[0174] The modified fibroin fiber (filament) according to the present embodiment is obtained
by spinning the above modified fibroin, and includes the above modified fibroin as
a main component. The modified fibroin fiber can be manufactured by a known spinning
method. That is, for example, when a modified fibroin fiber including a modified fibroin
as a main component is manufactured, first, the modified fibroin manufactured according
to the above method is added to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide
(DMF), formic acid, or hexafluoroisopropanol (HFIP), and as necessary, an inorganic
salt such as a dissolution promoting agent is added, and dissolved to prepare a dope
solution. Then, using this dope solution, spinning is performed by a known spinning
method such as wet spinning, dry spinning, dry wet spinning or melt spinning, and
thereby a modified fibroin fiber can be obtained. Examples of preferable spinning
methods include a wet spinning method and a dry wet spinning method.
[0175] FIG. 6 is an explanatory diagram schematically showing an example of a spinning device
for manufacturing a modified fibroin fiber. A spinning device 10 shown in FIG. 6 is
an example of a spinning device for dry wet spinning, and includes an extrusion device
1, an unstretched yarn manufacturing device 2, a wet heat stretching device 3, and
a drying device 4.
[0176] A spinning method using the spinning device 10 will be described. First, a dope solution
6 stored in a storage tank 7 is extruded from a cap 9 by a gear pump 8. In the laboratory
scale, the dope solution may be filled in a cylinder and extruded from a nozzle using
a syringe pump. Then, the extruded dope solution 6 is supplied to a coagulating liquid
11 in a coagulating liquid tank 20 through an air gap 19, the solvent is removed,
the modified fibroin is coagulated, and a fibrous coagulated component is formed.
Next, the fibrous coagulated component is supplied to hot water 12 in a stretched
bathtub 21 and stretched. The draw ratio is determined by a speed ratio between a
supply nip roller 13 and a take-off nip roller 14. Then, the stretched fibrous coagulated
component is supplied to the drying device 4 and dried in a yarn passage 22, and a
modified fibroin fiber 36 is obtained as a yarn wound component 5. 18a to 18g are
yarn guides.
[0177] The coagulating liquid 11 may be any solution in which a solvent can be removed,
and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol,
ethanol and 2-propanol, and acetone. The coagulating liquid 11 may appropriately contain
water. The temperature of the coagulating liquid 11 is preferably 0 to 30°C. When
a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the cap
9, the extrusion rate per hole is preferably 0.2 to 6.0 ml/hr and more preferably
1.4 to 4.0 ml/hr. The distance that the coagulated modified fibroin passes through
the coagulating liquid 11 (substantially, a distance from the yarn guide 18a to the
yarn guide 18b) may be any length at which the solvent can be efficiently removed,
and is, for example, 200 to 500 mm. The take-up speed of the unstretched yarn may
be, for example, 1 to 20 m/min, and is preferably 1 to 3 m/min. A time for which the
yarn is retained in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes,
and is preferably 0.05 to 0.15 minutes. In addition, stretching (pre-stretching) may
be performed in the coagulating liquid 11. The coagulating liquid tank 20 may be provided
in multiple stages, and stretching may be performed in each stage or a specific stage
as necessary.
[0178] Here, regarding the stretching performed when the modified fibroin fiber is obtained,
for example, in addition to pre-stretching performed in the coagulating liquid tank
20 and wet heat stretching performed in the stretched bathtub 21 described above,
dry heat stretching is also used.
[0179] The wet heat stretching can be performed in hot water, in a solution obtained by
adding an organic solvent and the like to hot water, or during steam heating. The
temperature may be, for example, 50 to 90°C, and is preferably 75 to 85°C. In the
wet heat stretching, for example, an unstretched yarn (or pre-stretched yarn) can
be stretched 1 to 10 times, and preferably stretched 2 to 8 times.
[0180] Dry heat stretching can be performed using an electric tubular furnace, a dry hot
plate, or the like. The temperature may be, for example, 140°C to 270°C, and is preferably
160°C to 230°C. In the dry heat stretching, the unstretched yarn (or pre-stretched
yarn) can be stretched, for example, 0.5 to 8 times, and preferably stretched 1 to
4 times.
[0181] The wet heat stretching and the dry heat stretching may be performed alone or in
multiple stages, or in combination. That is, the wet heat stretching and the dry heat
stretching can be appropriately performed in combination, for example, first stage
stretching is performed by wet heat stretching and second stage stretching is performed
by dry heat stretching or first stage stretching is performed by wet heat stretching,
second stage stretching is performed by wet heat stretching, and additionally third
stage stretching is performed by dry heat stretching.
[0182] The lower limit value of the final draw ratio is preferably more than 1 time, 2 times
or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times
or more, 8 times or more, or 9 times or more that of the unstretched yarn (or pre-stretched
yarn), and the upper limit value is preferably 40 times or less, 30 times or less,
20 times or less, 15 times or less, 14 times or less, 13 times or less, 12 times or
less, 11 times or less, or 10 times or less that of the unstretched yarn (or pre-stretched
yarn).
<Method for manufacturing artificial fibroin spun yarn>
[0183] An artificial fibroin spun yarn can be obtained by a method including a cutting process
in which the above modified fibroin fiber (filament) is cut into an appropriate length
to obtain a modified fibroin staple and a spinning process in which the obtained modified
fibroin staple is spun.
[0184] The cutting process can be performed using any device that can cut the modified fibroin
fiber. Examples of such a device include a desktop fiber cutting machine (s/NO.
IT-160201-NP-300).
[0185] The length of the modified fibroin staple is not particularly limited, and may be,
for example, 20 mm or more, 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.
[0186] The spinning process can be performed by a known spinning method. Examples of spinning
methods include a cotton spinning method, a worsted spinning method and a woolen spinning
method. A device used for these spinning methods is not particularly limited, and
a commonly used device can be used. In addition, in the spinning process, first, the
modified fibroin staple may be opened or defibrated by a fiber-opening machine (opener),
a defibrating machine (breaker) or the like.
[0187] The method for manufacturing an artificial fibroin spun yarn may include a crimping
process before or after the cutting process. The crimping process may be, for example,
a process in which a mechanical crimping method such as an indentation method is performed
or may be a process in which a modified fibroin fiber (filament) or a modified fibroin
staple is brought into contact with an aqueous medium and crimped (hereinafter referred
to as "water crimp" in some cases).
[0188] The aqueous medium is a liquid medium containing water (including water vapor) or
a gas (steam) medium. The aqueous medium may be water or a liquid in which water and
a hydrophilic solvent are mixed. In addition, regarding the hydrophilic solvent, for
example, a volatile solvent such as ethanol and methanol or vapors thereof can be
used. The aqueous medium may be a liquid in which water and a volatile solvent such
as ethanol and methanol are mixed and is preferably water or a liquid in which water
and ethanol are mixed. When an aqueous medium containing a volatile solvent or vapors
thereof is used, it is possible to increase a drying speed after water crimping, and
additionally, it is possible to impart soft texture to the finally obtained crimped
staple. The ratio between water and the volatile solvent or vapors thereof is not
particularly limited, and for example, the mass ratio of water:volatile solvent or
vapors thereof may be 10:90 to 90:10. The ratio of water is preferably 30 mass% or
more, and may be 40 mass% or 50 mass% or more.
[0189] The aqueous medium is preferably a liquid or gas containing water (including water
vapor) at 10 to 230°C. The temperature of the aqueous medium may be 10°C or higher,
25°C or higher, 40°C or higher, 60°C or higher, or 100°C or higher, and may be 230°C
or lower, 120°C or lower, or 100°C or lower. More specifically, when the aqueous medium
is a gas (steam), the temperature of the aqueous medium is preferably 100 to 230°C,
and more preferably 100 to 120°C. When the steam of the aqueous medium is at 230°C
or lower, it is possible to prevent denaturation of the modified fibroin filament
by heat. When the aqueous medium is a liquid, the temperature of the aqueous medium
is preferably 10°C or higher, 25°C or higher, or 40°C or higher in order to efficiently
impart crimp, and is preferably 60°C or lower in order to keep the fiber strength
of the modified fibroin filament high.
[0190] A time for which the aqueous medium is in contact is not particularly limited, and
may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and is preferably
10 minutes or shorter in consideration of the productivity. In addition, in the case
of steam, it is considered that a large shrinkage rate is obtained in a shorter time
than in the case of a liquid. The contact with the aqueous medium may be performed
under an atmospheric pressure or under a reduced pressure (for example, vacuum).
[0191] Examples of a method for contacting with an aqueous medium include a method for immersing
a modified fibroin filament or a modified fibroin staple in an aqueous medium, a method
for spraying aqueous medium steam to a modified fibroin filament or a modified fibroin
staple, and a method for exposing a modified fibroin filament or a modified fibroin
staple to an environment filled with aqueous medium steam. When the aqueous medium
is steam, the contact of the aqueous medium with the modified fibroin filament or
the modified fibroin staple can be performed using a general steam setting device.
Specific examples of steam setting devices include devices such as product name: FMSA
type steam setter-(commercially available from Fukushin Kougyo Co., Ltd.), and product
name: EPS-400 (commercially available from Tsujii Dyeing Machine Manufacturing Co.,
Ltd.). Specific examples of a method for crimping a modified fibroin filament or a
modified fibroin staple with aqueous medium steam include a method in which a modified
fibroin filament or a modified fibroin staple is accommodated in a predetermined accommodating
chamber, aqueous medium steam is introduced into the accommodating chamber, the temperature
in the accommodating chamber is adjusted to the above predetermined temperature (for
example, 100°C to 230°C), and steam is brought into contact with the modified fibroin
filament or the modified fibroin staple.
[0192] Here, the process of crimping the modified fibroin filament or the modified fibroin
staple by contact with an aqueous medium is performed preferably in a state in which
no tensile force is applied to single fibers and bundles (hanks, etc.) of the modified
fibroin filament and the modified fibroin staple (no tension is applied in the fiber
axis direction) or in a state in which only a predetermined degree is applied (a predetermined
amount of tension is applied in the fiber axis direction). When a tensile force applied
to the modified fibroin filament or the modified fibroin staple in this case is adjusted,
it is possible to control the degree of crimping. Examples of a method for preparing
a tensile force applied to the modified fibroin filament and the modified fibroin
staple include a method for adjusting a load applied to the filament and the staple
by hanging weights with various weights on the modified fibroin filament and the modified
fibroin staple, a method for fixing both ends while the filament and the staple are
slackened and changing the amount of slackness variously, and a method for winding
the filament around a winding target object such as a paper tube or a bobbin, and
appropriately changing a winding force (a tightening force on the paper tube or the
bobbin) in this case.
[0193] The modified fibroin filament or the modified fibroin staple is brought into contact
with the aqueous medium and may be then additionally dried. The drying method is not
particularly limited, and the drying may be performed by natural drying, or the drying
may be performed with hot air or a hot roller. The drying temperature is not particularly
limited, and may be for example, 20 to 150°C, and is preferably 40 to 120°C, and more
preferably 60 to 100°C.
[0194] Here, in order to further increase the shrinkage rate of the artificial fibroin spun
yarn, it is preferable not to perform the crimping process (particularly a water crimping
process). In addition, even if the crimping process is performed, when spinning is
performed using a staple in which the crimped staple and the uncrimped staple are
mixed in the spinning process, it is possible to further increase the shrinkage rate
of the artificial fibroin spun yarn.
[0195] The artificial fibroin spun yarn may be a single yarn or a mixed yarn such as a twin
yarn. Examples of artificial fibroin spun yarns include a spun yarn composed of 100%
modified fibroin, a mixed yarn composed of a staple of 100% modified fibroin and at
least one selected from among other protein staples, chemical fiber staples and the
like, a spun yarn using a staple containing a modified fibroin and other components,
and a mixed yarn composed of a staple containing a modified fibroin and other components
and at least one selected from among other protein staples and chemical fiber staples.
[0196] In order to make the modified fibroin staple easily loosen, an oil agent may be attached
in advance before the spinning process. The oil agent can be attached in any stage
in the manufacturing process. For example, the oil agent may be attached before the
cutting process, at the same time as the cutting process, or after the cutting process.
The oil agent is not particularly limited, and any of known oil agents that are used
for general purposes for imparting processability and functionality such as for imparting
an antistatic property, for reducing friction, for imparting flexibility, or imparting
water repellency can be used.
<Artificial fibroin spun yarn>
[0197] An artificial fibroin spun yarn according to the present embodiment includes the
above modified fibroin and is highly shrinkable when it is simply in contact with
water (water, water vapor, etc.). The term "highly shrinkable" means that the shrinkage
rate defined in the following Formula II is more than 1%.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0009)
[0198] The artificial fibroin spun yarn according to the present embodiment includes the
above modified fibroin, and has a shrinkage rate of greater than 1% defined in Formula
II, and thus can shrink at a higher shrinkage rate than protein spun yarns known in
the related art, and can shrink at a higher shrinkage rate under moderate conditions
than spun yarns composed of synthetic fibers known in the related art.
[0199] The shrinkage rate of the artificial fibroin spun yarn according to the present embodiment
may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8%
or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or
more. The upper limit of the shrinkage rate is not particularly limited, and may be
80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20%
or less.
[0200] In addition, the artificial fibroin spun yarn according to the present embodiment
includes the modified fibroin fiber (which means a filament after spinning) as a raw
material, and is highly shrinkable when there is no history of contact with water
(water, water vapor, etc.). That is, when there is no history of contact with water,
the shrinkage rate of the artificial fibroin spun yarn according to the present embodiment
may be, for example, more than 7%, and is preferably 15% or more, more preferably
more than 25%, still more preferably 32% or more, still more preferably 40% or more,
still more preferably 48% or more, particularly preferably 56% or more, more particularly
preferably 64% or more, and most preferably 72% or more. The upper limit of the shrinkage
rate is generally 80% or less.
[0201] The limit oxygen index (LOI) value of the artificial fibroin spun yarn according
to the present embodiment may be 18 or more, 20 or more, 22 or more, 24 or more, 26
or more, 28 or more, 29 or more, or 30 or more.
[0202] The artificial fibroin spun yarn according to the present embodiment has a maximum
hygroscopic heat generation of greater than 0.025°C/g determined according to the
following Formula A.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0010)
[0203] Here, in Formula A, the low humidity environment means an environment of a temperature
of 20°C and a relative humidity of 40%, and the high humidity environment means an
environment of a temperature of 20°C and a relative humidity of 90%.
[0204] The maximum hygroscopic heat generation of the artificial fibroin spun yarn according
to the present embodiment may be 0.026°C/g or more, 0.027°C/g or more, 0.028°C/g or
more, 0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, or 0.040°C/g or more.
The upper limit of the maximum hygroscopic heat generation is not particularly limited,
and is generally 0.060°C/g or less.
[0205] The heat retention index of the artificial fibroin spun yarn according to the present
embodiment determined according to the following Formula B may be 0.20 or more.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0011)
[0206] The heat retention index of the artificial fibroin spun yarn according to the present
embodiment may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or
more, or 0.32 or more. The upper limit of the heat retention index is not particularly
limited, and may be, for example, 0.60 or less or 0.40 or less.
(Shrinkage processing)
[0207] The shrinkage processing includes bringing an artificial fibroin spun yarn into contact
with water having a temperature lower than a boiling point (hereinafter referred to
as a "contact step"). The shrinkage processing may further include drying of the artificial
fibroin spun yarn after contact with water (after the contact step) (hereinafter referred
to as a "drying step").
[0208] The temperature of water in contact with the artificial fibroin spun yarn in the
contact step may be lower than the boiling point. Thereby, handling properties and
workability of shrinkage processing and the like are improved. In addition, in order
to sufficiently shorten the shrinking time, the lower limit value of the temperature
of water is preferably 10°C or higher, more preferably 40°C or higher, and still more
preferably 70°C or higher. The upper limit value of the temperature of water is preferably
90°C or lower.
[0209] In the contact step, a method for bringing water into contact with the artificial
fibroin spun yarn is not particularly limited. Examples of the method include a method
for immersing an artificial fibroin spun yarn in water, a method for spraying water
at room temperature or in a heated state such as steam to an artificial fibroin spun
yarn, and a method for exposing an artificial fibroin spun yarn to a high humidity
environment filled with water vapor. Among these methods, in the contact step, a method
for immersing an artificial fibroin spun yarn in water is preferable because the shrinking
time can be effectively shortened and processing facilities can be simplified.
[0210] The drying step is a step of drying the artificial fibroin spun yarn that has undergone
the contact step. For example, the drying may be performed by natural drying or may
be forcibly performed by using a drying device. Regarding the drying device, any known
contact type or non-contact type drying device can be used. In addition, the drying
temperature is not particularly limited as long as the temperature is, for example,
lower than a temperature at which proteins (modified fibroin, etc.) contained in the
artificial fibroin spun yarn are decompose and the artificial fibroin spun yarn is
thermally damaged, and is generally a temperature in a range of 20 to 150°C and preferably
a temperature in a range of 50 to 100°C. When the temperature is in this range, the
artificial fibroin spun yarn is more quickly and efficiently dried without causing
thermal damage to the artificial fibroin spun yarn or decomposition of proteins contained
in the artificial fibroin spun yarn. The drying time is appropriately set according
to the drying temperature and the like, and for example, a time at which the influence
of overdrying on the quality, physical properties, and the like of the artificial
fibroin spun yarn can be eliminated as much as possible is used.
[0211] The shrinkage rate of the artificial fibroin spun yarn according to the present embodiment
can be controlled by controlling, for example, the temperature of water in contact,
the contact time with water, and the tensile force when brought into contact with
water.
<High-shrinkage artificial fibroin spun yarn and method for manufacturing the same>
[0212] Since the artificial fibroin spun yarn according to the present embodiment is highly
shrinkable, for example, the high-shrinkage artificial fibroin spun yarn can be obtained
through the above shrinkage processing (the contact step, and as necessary the drying
step).
[0213] In the high-shrinkage artificial fibroin spun yarn according to the present embodiment,
the shrinkage rate defined by the following Formula I is more than 1%.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0012)
[0214] The shrinkage rate of the high-shrinkage artificial fibroin spun yarn according to
the present embodiment may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or
more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25%
or more, or 30% or more. The upper limit of the shrinkage rate is not particularly
limited, and may be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less,
30% or less, or 20% or less.
[0215] In addition, the artificial fibroin spun yarn according to the present embodiment
includes the modified fibroin fiber (which means a filament after spinning) as a raw
material, and is highly shrinkable when there is no history of contact with water
(water, water vapor, etc.). That is, when there is no history of contact with water,
the shrinkage rate of the high-shrinkage artificial fibroin spun yarn according to
the present embodiment may be, for example, more than 7%, and is preferably 15% or
more, more preferably more than 25%, still more preferably 32% or more, still more
preferably 40% or more, still more preferably 48% or more, particularly preferably
56% or more, more particularly preferably 64% or more, and most preferably 72% or
more. The upper limit of the shrinkage rate is generally 80% or less.
[0216] The limit oxygen index (LOI) value of the artificial fibroin spun yarn according
to the present embodiment may be 18 or more, 20 or more, 22 or more, 24 or more, 26
or more, 28 or more, 29 or more, or 30 or more.
[0217] The maximum hygroscopic heat generation of the artificial fibroin spun yarn according
to the present embodiment determined according to the following Formula A may be greater
than 0.025°C/g.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0013)
[0218] Here, in Formula A, the low humidity environment means an environment of a temperature
of 20°C and a relative humidity of 40%, and the high humidity environment means an
environment of a temperature of 20°C and a relative humidity of 90%.
[0219] The maximum hygroscopic heat generation of the artificial fibroin spun yarn according
to the present embodiment may be 0.026°C/g or more, 0.027°C/g or more, 0.028°C/g or
more, 0.029°C/g or more, 0.030°C/g or more, 0.035°C/g or more, or 0.040°C/g or more.
The upper limit of the maximum hygroscopic heat generation is not particularly limited,
and is generally 0.060°C/g or less.
[0220] The heat retention index of the artificial fibroin spun yarn according to the present
embodiment determined according to the following Formula B may be 0.20 or more.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0014)
[0221] The heat retention index of the artificial fibroin spun yarn according to the present
embodiment may be 0.22 or more, 0.24 or more, 0.26 or more, 0.28 or more, 0.30 or
more, or 0.32 or more. The upper limit of the heat retention index is not particularly
limited, and may be, for example, 0.60 or less or 0.40 or less.
[Method for shrinking artificial fibroin spun yarn]
[0222] A method for shrinking an artificial fibroin spun yarn according to the present embodiment
includes a process of bringing an artificial fibroin spun yarn including a modified
fibroin into contact with water having a temperature lower than a boiling point and
performing shrinking. The shrinkage process may further include drying of the artificial
fibroin spun yarn after contact with water. That is, in the same manner as in the
above shrinkage processing, the method for shrinking an artificial fibroin spun yarn
according to the present embodiment can be performed.
Examples
[0223] The present invention will be described below in more detail with reference to examples
and the like. However, the present invention is not limited to the following examples.
[Test Example 1: manufacture of modified fibroin (modified spider silk fibroin)]
(1) Production of modified fibroin expression vector
[0224] A modified fibroin (PRT399) having an amino acid sequence shown in SEQ ID NO 18,
a modified fibroin (PRT380) having an amino acid sequence shown in SEQ ID NO 12, a
modified fibroin (PRT410) having an amino acid sequence shown in SEQ ID NO 13, a modified
fibroin (PRT799) having an amino acid sequence shown in SEQ ID NO 15, and a modified
fibroin (PRT918) having an amino acid sequence shown in SEQ ID NO 37 were designed.
[0225] Nucleic acids that encode a protein having the designed amino acid sequence were
synthesized. In the nucleic acids, an NdeI site was added to the 5' end and an EcoRI
site was added downstream of the stop codon. These five nucleic acids were cloned
into cloning vectors (pUC118). Then, the same nucleic acid was treated with restriction
enzymes NdeI and EcoRI and cut, and then recombined into a protein expression vector
pET-22b(+) to obtain an expression vector.
(2) Expression of modified fibroin
[0226] E. coli BLR (DE3) was transformed with the obtained expression vector. The transformed
E. coli was cultured in a 2 mL LB culture medium containing ampicillin for 15 hours. The
culture solution was added to a 100 mL culture medium containing ampicillin for seed
culturing (Table 4) so that the OD
600 became 0.005. The temperature of the culture solution was maintained at 30°C and
flask culture was performed until the OD
600 became 5 (about 15 hours), and thereby a seed culture solution was obtained.
[Table 4]
Culture medium for seed culturing |
Sample |
Concentration (g/L) |
Glucose |
5.0 |
KH2PO4 |
4.0 |
K2HPO4 |
9.3 |
Yeast Extract |
6.0 |
Ampicillin |
0.1 |
[0227] The seed culture solution was added to a jar fermenter containing a 500 ml production
culture medium (Table 5) so that the OD
600 became 0.05. The temperature of the culture solution was maintained at 37°C and the
culture was performed under control with a constant pH of 6.9. In addition, the dissolved
oxygen concentration in the culture solution was maintained at 20% of the dissolved
saturated oxygen concentration.
[Table 5]
Production culture medium |
Sample |
Concentration (g/L) |
Glucose |
12.0 |
KH2PO4 |
9.0 |
MgSO4·7H2O |
2.4 |
Yeast Extract |
15 |
FeSO4·7H2O |
0.04 |
MnSO4·5H2O |
0.04 |
CaCl2·2H2O |
0.04 |
ADEKA NOL (ADEKA, LG-295S) |
0.1 (mL/L) |
[0228] Immediately after glucose in the production culture medium was completely consumed,
a feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of
1 mL/min. The temperature of the culture solution was maintained at 37°C and the culture
was performed under control with a constant pH of 6.9. The culture was performed for
20 hours while maintaining the dissolved oxygen concentration in the culture solution
at 20% of the dissolved saturated oxygen concentration. Then, 1 M isopropyl-β-thiogalactopyranoside
(IPTG) was added to the culture solution so that the final concentration was 1 mM,
and thereby a desired modified fibroin was expressed and induced. When 20 hours had
elapsed after IPTG was added, the culture solution was centrifuged, and bacteria were
collected. SDS-PAGE was performed using bacteria prepared from the culture solution
before IPTG was added and after IPTG was added, and the expression of the desired
modified fibroin was confirmed according to the appearance of a band with a size corresponding
to the desired modified fibroin depending on the addition of IPTG.
(3) Purification of modified fibroin
[0229] The bacteria collected 2 hours after IPTG was added were washed with a 20 mM Tris-HCl
buffer (pH 7.4). The bacteria after washing were suspended in a 20 mM Tris-HCl buffer
solution (pH 7.4) containing about 1 mM PMSF, and cells were crushed using a high
pressure homogenizer (commercially available from GEA Niro Soavi). The crushed cells
were centrifuged to obtain a precipitate. The obtained precipitate was washed with
a 20 mM Tris-HCl buffer solution (pH 7.4) until it became highly pure. The precipitate
after washing was suspended in a 8 M guanidine buffer (8 M guanidine hydrochloride,
10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the
concentration became 100 mg/mL, and stirred with a stirrer at 60°C for 30 minutes
and dissolved. After the dissolution, dialysis was performed with water using a dialysis
tube (cellulose tube 36/32 commercially available from Sanko Junyaku Co., Ltd.). White
aggregated proteins obtained after dialysis were collected through centrifugation.
Water was removed from the collected aggregated proteins in a freeze dryer, and thereby
lyophilized powders of desired modified fibroins (PRT399, PRT380, PRT410, PRT799 and
PRT918) were obtained.
[Reference Example 1: manufacture of modified fibroin fiber (filament) and evaluation
of shrinkability]
[0230] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass%
was prepared as a solvent, and the lyophilized powder of the modified fibroin (PRT399,
PRT380, PRT410 or PRT799) obtained in Test Example 1 was added thereto at a concentration
of 18 mass% or 24 mass% (refer to Table 6), and dissolved using a shaker for 3 hours.
Then, insoluble substances and bubbles were removed to obtain a modified fibroin solution.
[0231] The obtained modified fibroin solution was used as a dope solution (spinning stock
solution), and a spun and stretched modified fibroin fiber (filament) was manufactured
by dry wet spinning using a spinning device similar to the spinning device 10 shown
in FIG. 6. The spinning device that was used was obtained by additionally providing
a second unstretched yarn manufacturing device (a second bath) between the unstretched
yarn manufacturing device 2 (a first bath) and the wet heat stretching device 3 (a
third bath) in the spinning device 10 shown in FIG. 6. The dry wet spinning conditions
were as follows.
Extrusion nozzle diameter: 0.2 mm
[0232] Liquids and temperatures in the first bath to the third bath: refer to Table 6
Total draw ratio: refer to Table 6
Drying temperature: 60°C
[Table 6]
|
Dope solution |
First bath |
Second bath |
Third bath |
Total draw ratio (times) |
Modified Concentration fibroin (mass%) |
Liquid |
Temperature (°C) |
Liquid Temperature (°C) |
Liquid |
Temperature (°C) |
Manufacturing Example 1 |
PRT799 |
24 |
100% methanol |
-5 |
100% methanol |
16 |
Water |
17 |
1 |
Manufacturing Example 2 |
|
|
|
|
|
|
|
|
2 |
Manufacturing Example 3 |
|
|
|
|
|
|
|
|
3 |
Manufacturing Example 4 |
|
|
|
|
|
|
|
|
4 |
Manufacturing Example 5 |
|
18 |
|
|
|
|
|
|
1 |
Manufacturing Example 6 |
|
|
|
|
|
|
|
|
2 |
Manufacturing Example 7 |
|
|
|
|
|
|
|
|
3 |
Manufacturing Example 8 |
|
|
|
|
|
|
|
|
4 |
Manufacturing Example 9 |
PRT410 |
24 |
|
-11 |
|
14 |
|
|
1 |
Manufacturing Example 10 |
|
|
|
|
|
|
|
|
2 |
Manufacturing Example 11 |
|
|
|
|
|
|
|
|
3 |
Manufacturing Example 12 |
|
|
|
|
|
|
|
|
4 |
Manufacturing Example 13 |
PRT399 |
|
|
|
|
|
|
|
1 |
Manufacturing Example 14 |
|
|
|
|
|
|
|
|
2 |
Manufacturing Example 15 |
|
|
|
|
|
|
|
|
3 |
Manufacturing Example 16 |
PRT380 |
|
|
|
|
11 |
|
|
1 |
Manufacturing Example 17 |
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2 |
Manufacturing Example 18 |
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3 |
Manufacturing Example 19 |
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4 |
(Evaluation of shrinkability)
[0233] The shrinkage rates of the modified fibroin fibers (filaments) obtained in Manufacturing
Examples 1 to 19 were evaluated. That is, regarding the modified fibroin fibers, a
shrinkage rate when shrinkage processing 1 including being brought into contact with
water having a temperature lower than a boiling point was performed (hereinafter referred
to as a "primary shrinkage rate"), and a shrinkage rate when shrinkage processing
2 including performing drying at room temperature after bringing into contact with
water having a temperature lower than a boiling point were performed (hereinafter
referred to as a "secondary shrinkage rate") were evaluated.
<Primary shrinkage rate>
[0234] A plurality of modified fibroin fibers for testing, each having a length of 30 cm,
were cut out from a wound product of the modified fibroin fibers obtained in Manufacturing
Examples 1 to 19. The plurality of modified fibroin fibers were bundled to obtain
a modified fibroin fiber bundle having a fineness of 150 denier. A 0.8 g plumb bob
was attached to each modified fibroin fiber bundle, and in that state, each modified
fibroin fiber bundle was immersed in water at a temperature shown in Tables 7 to 10
for 10 minutes (the shrinkage processing 1). Then, the length of each modified fibroin
fiber bundle was measured in water. The length of the modified fibroin fiber bundle
in water was measured while the 0.8 g plumb bob was attached to the modified fibroin
fiber bundle in order to eliminate curling of the modified fibroin fiber bundle. Then,
the primary shrinkage rate (%) of each modified fibroin fiber was calculated according
to the following Formula III. In Formula III, L0 represents the length (here, 30 cm)
of the modified fibroin fiber bundle before shrinkage processing was performed, and
Lw represents the length of the modified fibroin fiber bundle subjected to the shrinkage
processing 1.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0015)
<Secondary shrinkage rate>
[0235] After immersion in water for evaluation of the primary shrinkage rate, the modified
fibroin fiber bundle was removed from water. The removed modified fibroin fiber bundle
with the 0.8 g plumb bob attached thereto was dried at room temperature for 2 hours
(the shrinkage processing 2). After the drying, the length of each modified fibroin
fiber bundle was measured. Then, the secondary shrinkage rate (%) of each modified
fibroin fiber was calculated according to the following Formula IV. In Formula IV,
L0 represents the length (here, 30 cm) of the modified fibroin fiber bundle before
shrinkage processing was performed, and Lwd represents the length of the modified
fibroin fiber bundle subjected to the shrinkage processing 2.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0016)
[0236] The results are shown in Tables 7 to 10.
[Table 7]
Modified fibroin fiber |
Temperature of water lower than boiling point (°C) |
Primary shrinkage rate (%) |
Secondary shrinkage rate (%) |
Manufacturing Example 1 |
24 wt% PRT799 x1 |
20 |
0.0 |
7.8 |
Manufacturing Example 2 |
24 wt% PRT799 x2 |
|
-1.2 |
10.3 |
Manufacturing Example 3 |
24 wt% PRT799 x3 |
|
7.2 |
21.2 |
Manufacturing Example 4 |
24 wt% PRT799 x4 |
|
13.5 |
26.3 |
Manufacturing Example 6 |
18 wt% PRT799 x2 |
|
-2.3 |
9.5 |
Manufacturing Example 7 |
18 wt% PRT799 x3 |
|
6.0 |
19.7 |
Manufacturing Example 8 |
18 wt% PRT799 x4 |
|
14.3 |
27.5 |
Manufacturing Example 2 |
24 wt% PRT799 x2 |
40 |
-5.3 |
7.2 |
Manufacturing Example 3 |
24 wt% PRT799 x3 |
|
8.7 |
21.3 |
Manufacturing Example 4 |
24 wt% PRT799 x4 |
|
14.5 |
26.0 |
Manufacturing Example 6 |
18 wt% PRT799 x2 |
|
-4.3 |
7.3 |
Manufacturing Example 7 |
18 wt% PRT799 x3 |
|
6.2 |
18.3 |
Manufacturing Example 8 |
18 wt% PRT799 x4 |
|
16.0 |
28.7 |
Manufacturing Example 3 |
24 wt% PRT799 x3 |
60 |
6.8 |
21.0 |
Manufacturing Example 4 |
24 wt% PRT799 x4 |
|
15.0 |
27.5 |
Manufacturing Example 6 |
18 wt% PRT799 x2 |
|
-1.5 |
10.7 |
Manufacturing Example 7 |
18 wt% PRT799 x3 |
|
3.3 |
18.2 |
Manufacturing Example 8 |
18 wt% PRT799 x4 |
|
16.2 |
29.0 |
[Table 8]
Modified fibroin fiber |
Temperature of water lower than boiling point (°C) |
Primary shrinkage rate (%) |
Secondary shrinkage rate (%) |
Manufacturing Example 10 |
24 wt% PRT410 x2 |
20 |
-2.3 |
8.7 |
Manufacturing Example 11 |
24 wt% PRT410 x3 |
4.7 |
16.7 |
Manufacturing Example 12 |
24 wt% PRT410 x4 |
10.3 |
22.3 |
Manufacturing Example 11 |
24 wt% PRT410 x3 |
40 |
4.7 |
17.5 |
Manufacturing Example 12 |
24 wt% PRT410 x4 |
11.5 |
24.0 |
Manufacturing Example 11 |
24 wt% PRT410 x3 |
60 |
2.0 |
16.5 |
Manufacturing Example 12 |
24 wt% PRT410 x4 |
10.8 |
25.0 |
[Table 9]
Modified fibroin fiber |
Temperature of water lower than boiling point (°C) |
Primary shrinkage rate (%) |
Secondary shrinkage rate (%) |
Manufacturing Example 13 |
24 wt% PRT399 x1 |
20 |
-3.5 |
7.6 |
Manufacturing Example 14 |
24 wt% PRT399 x2 |
3.7 |
12.5 |
Manufacturing Example 15 |
24 wt% PRT399 x3 |
7.0 |
16.8 |
Manufacturing Example 14 |
24 wt% PRT399 x2 |
40 |
3.0 |
12.7 |
Manufacturing Example 15 |
24 wt% PRT399 x3 |
7.3 |
16.7 |
Manufacturing Example 14 |
24 wt% PRT399 x2 |
60 |
3.3 |
9.3 |
Manufacturing Example 15 |
24 wt% PRT399 x3 |
6.8 |
14.2 |
[Table 10]
Modified fibroin fiber |
Temperature of water lower than boiling point (°C) |
Primary shrinkage rate (%) |
Secondary shrinkage rate (%) |
Manufacturing Example 16 |
24 wt% PRT380 x1 |
20 |
-1.1 |
9.4 |
Manufacturing Example 17 |
24 wt% PRT380 x2 |
2.7 |
13.3 |
Manufacturing Example 18 |
24 wt% PRT380 x3 |
7.0 |
17.7 |
Manufacturing Example 19 |
24 wt% PRT380 x4 |
10.0 |
20.2 |
Manufacturing Example 17 |
24 wt% PRT380 x2 |
40 |
3.3 |
14.2 |
Manufacturing Example 18 |
24 wt% PRT380 x3 |
7.7 |
19.0 |
Manufacturing Example 19 |
24 wt% PRT380 x4 |
12.0 |
22.0 |
Manufacturing Example 17 |
24 wt% PRT380 x2 |
60 |
2.7 |
14.3 |
Manufacturing Example 18 |
24 wt% PRT380 x3 |
8.2 |
20.3 |
Manufacturing Example 19 |
24 wt% PRT380 x4 |
12.0 |
23.2 |
[0237] Based on the above results, it was found that the modified fibroin fiber (filament)
according to the present invention had a sufficiently high shrinkage rate. Therefore,
it was confirmed that the artificial fibroin spun yarn spun from the modified fibroin
fiber (filament) according to the present invention had a sufficiently high shrinkage
rate. In addition, it can be understood that the shrinkage rate of the modified fibroin
fiber (filament) and the artificial fibroin spun yarn according to the present invention
can be controlled by controlling, for example, the temperature of water in contact,
the contact time with water, and the tensile force when brought into contact with
water.
[Example 1: manufacture and evaluation of artificial fibroin spun yarn]
(1) Manufacture of modified fibroin fiber (filament)
[0238] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass%
was prepared as a solvent, and the lyophilized powder of the modified fibroin (PRT799)
obtained in Test Example 1 was added thereto at a concentration of 24 mass%, and dissolved
using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to
obtain a modified fibroin solution.
[0239] The obtained modified fibroin solution was used as a dope solution (spinning stock
solution), and dry wet spinning was performed using a spinning device similar to the
spinning device 10 shown in FIG. 6 to obtain modified fibroin filaments (24 multifilaments).
The obtained modified fibroin filaments were wound on a bobbin. The dry wet spinning
conditions were as follows.
Temperature of coagulating liquid (methanol): 5 to 10°C
Draw ratio: 5 times
Drying temperature: 80°C
(2) Manufacture of artificial fibroin spun yarn
[0240] The modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm
using a desktop fiber cutting machine to obtain a modified fibroin staple. A part
of the obtained staple was immersed in water at 40°C for 1 minute and curled and crimped
and then dried at 40°C for 18 hours to obtain a crimped staple. Then, a staple including
the crimped staple and the uncrimped staple at a ratio of 7:3 (weight ratio) was spun
using a known spinning device to obtain an artificial fibroin spun yarn 1. The fineness
(yarn number) of the artificial fibroin spun yarn 1 was 30 Nm.
(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)
[0241] The artificial fibroin spun yarn 1 was cut into a length of 60 cm and immersed in
water at 40°C for 1 minute and then dried with air. The length of the spun yarn dried
with air was measured, and the shrinkage rate was determined according to the following
Formula V. The results are shown in Table 11.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0017)
[0242] Here, the "length of the spun yarn before immersion in water" was 60 cm.
[Example 2: manufacture and evaluation of artificial fibroin spun]
(1) Manufacture of modified fibroin fiber (filament)
[0243] A modified fibroin filament was obtained in the same method as in Example 1.
(2) Manufacture of artificial fibroin spun yarn
[0244] The modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm
using a desktop fiber cutting machine to obtain a modified fibroin staple. The obtained
staple (uncrimped staple) was spun using a known spinning device to obtain an artificial
fibroin spun yarn 2. The fineness (yarn number) of the artificial fibroin spun yarn
2 was 48 Nm.
(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)
[0245] The shrinkage rate of the artificial fibroin spun yarn 2 was determined in the same
method as in Example 1. The results are shown in Table 11.
[Comparative Example 1: manufacture and evaluation of artificial fibroin spun yarn]
(1) Manufacture of modified fibroin fiber (filament)
[0246] A modified fibroin filament was manufactured in the same manner as in Example 1 except
that, in place of lyophilized powder of the modified fibroin (PRT799), lyophilized
powder in which the lyophilized powder of the modified fibroin (PRT918) and the lyophilized
powder of the modified fibroin (PRT799) obtained in Test Example 1 were mixed at a
ratio of 6:4 (weight ratio) was used.
(2) Manufacture of artificial fibroin spun yarn
[0247] The modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm
using a desktop fiber cutting machine to obtain a modified fibroin staple. The obtained
staple was immersed in water at 40°C for 1 minute and curled and crimped, and then
dried at 40°C for 18 hours to obtain a crimped staple. Then, the crimped staple was
spun using a known spinning device to obtain an artificial fibroin spun yarn 3. The
fineness (yarn number) of the artificial fibroin spun yarn 3 was 3 Nm.
(3) Evaluation of artificial fibroin spun yarn (water shrinkage test)
[0248] The shrinkage rate of the artificial fibroin spun yarn 3 was determined in the same
method as in Example 1. The results are shown in Table 11.
[0249] [Comparative Example 2: evaluation of PET spun yarn] For comparison, a single spun
yarn (PET spun yarn) composed of commercially available 100% PET and having a fineness
of 30 Nm was purchased. The water shrinkage test was performed in the same method
as in Example 1, and the shrinkage rate of the PET spun yarn was determined. The results
are shown in Table 11.
[Table 11]
|
Fineness (Nm) |
Single yarn/twin yarn |
Before shrinking (cm) |
After shrinking (cm) |
Shrinkage rate |
Note |
Example 1 |
30 |
Single yarn |
60 |
50 |
16.7% |
Crimped staple/uncrimped staple=7/3 |
Example 2 |
48 |
Twin yarn |
60 |
40.2 |
33.0% |
Uncrimped staple 100% |
Comparative Example 1 |
3 |
Single yarn |
60 |
59.4 |
1% |
Crimped staple 100% |
Comparative Example 2 |
30 |
Single yarn |
60 |
59.5 |
0.8% |
- |
[0250] The artificial fibroin spun yarn of the present invention had a sufficiently high
shrinkage rate and the high-shrinkage artificial fibroin spun yarn after shrinkage
had excellent tactile feel and flexibility. In addition, since the high-shrinkage
artificial fibroin spun yarn of the present invention could be manufactured by bringing
an artificial fibroin spun yarn into contact with water having a temperature lower
than a boiling point and as necessary performing drying after contact with water,
it could be manufactured safely.
[Test Example 2: manufacture of modified fibroin]
[0251] A modified fibroin (PRT918) having an amino acid sequence shown in SEQ ID NO 37,
a modified fibroin (PRT966) having an amino acid sequence shown in SEQ ID NO 40, and
a modified fibroin (PRT799) having an amino acid sequence shown in SEQ ID NO 15 were
designed. A nucleic acid that encodes the designed modified fibroins was synthesized.
In the nucleic acid, an NdeI site was added to the 5' end and an EcoRI site was added
downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118).
Then, the same nucleic acid was treated with restriction enzymes NdeI and EcoRI and
cut, and then recombined into a protein expression vector pET-22b(+) to obtain an
expression vector.
[0252] E. coli BLR (DE3) was transformed with the obtained expression vector. The transformed
E. coli was cultured in a 2 mL LB culture medium containing ampicillin for 15 hours. The
culture solution was added to a 100 mL culture medium containing ampicillin for seed
culturing (Table 12) so that the OD
600 became 0.005. The temperature of the culture solution was maintained at 30°C and
flask culture was performed until the OD
600 became 5 (about 15 hours), and thereby a seed culture solution was obtained.
[Table 12]
Culture medium for seed culturing |
Sample |
Concentration (g/L) |
Glucose |
5.0 |
KH2PO4 |
4.0 |
K2HPO4 |
9.3 |
Yeast Extract |
6.0 |
Ampicillin |
0.1 |
[0253] The seed culture solution was added to a jar fermenter containing a 500 mL production
culture medium (Table 13) so that the OD
600 became 0.05. The temperature of the culture solution was maintained at 37°C and the
culture was performed under control with a constant pH of 6.9. In addition, the dissolved
oxygen concentration in the culture solution was maintained at 20% of the dissolved
saturated oxygen concentration.
[Table 13]
Production culture medium |
Sample |
Concentration (g/L) |
Glucose |
12.0 |
KH2PO4 |
9.0 |
MgSO4·7H2O |
2.4 |
Yeast Extract |
15 |
FeSO4·7H2O |
0.04 |
MnSO4·5H2O |
0.04 |
CaCl2·2H2O |
0.04 |
ADEKA NOL (ADEKA, LG-295S) |
0.1 (mL/L) |
[0254] Immediately after glucose in the production culture medium was completely consumed,
a feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of
1 mL/min. The temperature of the culture solution was maintained at 37°C and the culture
was performed under control with a constant pH of 6.9. In addition, the dissolved
oxygen concentration in the culture solution was maintained at 20% of the dissolved
saturated oxygen concentration, and the culture was performed for 20 hours. Then,
1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution so
that the final concentration was 1 mM, and thereby a modified fibroin was expressed
and induced. When 20 hours had elapsed after IPTG was added, the culture solution
was centrifuged, and bacteria were collected. SDS-PAGE was performed using bacteria
prepared from the culture solution before IPTG was added and after IPTG was added,
and the expression of the desired modified fibroin was confirmed according to the
appearance of a band with a size corresponding to the desired modified fibroin depending
on the addition of IPTG.
[0255] The bacteria collected 2 hours after IPTG was added were washed with a 20 mM Tris-HCl
buffer (pH 7.4). The bacteria after washing were suspended in a 20 mM Tris-HCl buffer
solution (pH 7.4) containing about 1 mM PMSF, and cells were crushed using a high
pressure homogenizer (commercially available from GEA Niro Soavi). The crushed cells
were centrifuged to obtain a precipitate. The obtained precipitate was washed with
a 20 mM Tris-HCl buffer solution (pH 7.4) until it became highly pure. The precipitate
after washing was suspended in a 8 M guanidine buffer (8 M guanidine hydrochloride,
10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the
concentration became 100 mg/mL, and stirred with a stirrer at 60°C for 30 minutes
and dissolved. After the dissolution, dialysis was performed with water using a dialysis
tube (cellulose tube 36/32 commercially available from Sanko Junyaku Co., Ltd.). White
aggregated proteins obtained after dialysis were collected through centrifugation,
water was removed in a freeze dryer, and lyophilized powders were collected to obtain
modified fibroins (PRT918, PRT966 and PRT799).
[0256] PRT918 and PRT966 were hydrophobic modified fibroins having an average HI of greater
than 0. PRT799 was a hydrophilic modified fibroin having an average HI of 0 or less.
[Reference Example 2: evaluation of flame retardancy of modified fibroin]
[0257] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass%
was prepared as a solvent, and the lyophilized powder of the modified fibroin (PRT799)
obtained in Test Example 2 was added thereto at a concentration of 24 mass%, and dissolved
using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to
obtain a modified fibroin solution (spinning stock solution).
[0258] The prepared spinning stock solution was filtered at 90°C with a metal filter having
an opening of 5 µm, and then left in a 30 mL stainless syringe, bubbles were removed,
and then the solution was discharged from a solid nozzle having a needle diameter
of 0.2 mm to a 100 mass% methanol solidification tank. The discharge temperature was
90°C. After coagulation, the obtained raw yarn was wound and naturally dried to obtain
a modified fibroin fiber (raw fiber).
[0259] A knitted fabric was manufactured using a twisted yarn obtained by twisting the obtained
raw fibers according to circular knitting using a circular knitting machine. The knitted
fabric had a thickness of 180 denier and a gauge number of 18. 20 g was cut out from
the obtained knitted fabric to prepare a test piece.
[0260] The flammability test was performed according to a test method for powder granules
or a synthetic resin having a low melting point,
Fire Department Dangerous Goods Regulation Division Fire Department No. 50, May 31,
1995. The test was performed under conditions of a temperature of 22°C, a relative humidity
of 45%, and an atmospheric pressure of 1,021 hPa. Table 14 shows the measurement results
(oxygen concentration (%), combustion rate (%), and conversion combustion rate (%)).
[Table 14]
Oxygen concentration (%) |
Combustion rate (%) |
Conversion combustion rate (%) |
20.0 |
39.1 |
40.1 |
27.0 |
48.1 |
49.3 |
28.0 |
51.9 |
53.2 |
30.0 |
53.6 |
54.9 |
50.0 |
61.2 |
62.7 |
70.0 |
91.1 |
93.3 |
100.0 |
97.6 |
100.0 |
[0261] As a result of the flammability test, the limit oxygen index (LOI) value of the knitted
fabric knitted with the modified fibroin (PRT799) fiber was 27.2. Generally, when
the LOI value was 26 or more, it was considered that the component had a flame retardancy.
It was found that the modified fibroin had an excellent flame retardancy.
[Reference Example 3: Evaluation of hygroscopic heat generation characteristics of
modified fibroin]
[0262] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass%
was prepared as a solvent, and the lyophilized powder of the modified fibroin obtained
in Test Example 2 was added thereto at a concentration of 24 mass%, and dissolved
using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to
obtain a modified fibroin solution (spinning stock solution).
[0263] The prepared spinning stock solution was filtered at 60°C with a metal filter having
an opening of 5 µm, and then left in a 30 mL stainless syringe, bubbles were removed,
and then the solution was discharged from a solid nozzle having a needle diameter
of 0.2 mm to a 100 mass% methanol solidification tank. The discharge temperature was
60°. After coagulation, the obtained raw yarn was wound and naturally dried to obtain
a modified fibroin fiber (raw fiber).
[0264] For comparison, regarding raw fibers, commercially available wool fibers, cotton
fibers, Tencel fibers, rayon fibers and polyester fibers were prepared.
[0265] Knitted fabrics were manufactured using the raw fibers according to flat knitting
using a flat knitting machine. The knitted fabric obtained using the PRT918 fibers
as raw fibers had a thickness of 1/30 N (wool count single yarn) and a gauge number
of 18. The knitted fabric obtained using the PRT799 fibers as raw fibers had a thickness
of 1/30 N (wool count single yarn) and a gauge number of 16. The thickness and the
gauge number of the knitted fabric obtained using other raw fibers were adjusted such
that the cover factor was almost the same as that of the knitted fabric obtained using
the PRT918 fibers and the PRT799 fibers. The details were as follows.
Wool thickness: 2/30 N (twin yarn), gauge number: 14
Cotton thickness: 2/34 N (twin yarn), gauge number: 14
Tencel thickness: 2/30 N (twin yarn), gauge number: 15
Rayon thickness: 1/38 N (single yarn), gauge number: 14
Polyester thickness: 1/60 N (single yarn), gauge number: 14
[0266] Two pieces of knitted fabric cut into 10 cm×10 cm were combined, and four sides were
sewn together to prepare a test piece (sample). The test piece was left in a low humidity
environment (a temperature of 20±2°C and a relative humidity of 40±5%) for 4 hours
or longer, and then moved to a high humidity environment (a temperature of 20±2°C
and a relative humidity of 90±5%), and the temperature was measured using a temperature
sensor attached to the center inside the test piece for 30 minutes at 1-minute intervals.
[0267] The maximum hygroscopic heat generation was determined from the measurement results
according to the following Formula A.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0018)
[0268] FIG. 7 is a graph showing an example of results of a hygroscopic and exothermic test.
In the graph, the horizontal axis represents the time (min) for which the sample was
left in the high humidity environment when the time at which the sample was moved
from the low humidity environment to the high humidity environment was set as 0. In
the graph, the vertical axis represents the temperature measured using the temperature
sensor (sample temperature). In the graph shown in FIG. 7, the point indicated by
M corresponds to the maximum value of the sample temperature.
[0269] Table 15 shows calculation results of the maximum hygroscopic heat generation.
[Table 15]
Raw fiber |
Maximum hygroscopic heat generation (°C/g) |
PRT918 |
0.040 |
PRT799 |
0.031 |
Wool |
0.020 |
Cotton |
0.021 |
Tencel |
0.018 |
Rayon |
0.025 |
Polyester |
0.010 |
[0270] As shown in Table 15, it was found that the modified fibroin (PRT918 and PRT799)
had a higher maximum hygroscopic heat generation and a superior hygroscopic heat generation
characteristics than materials in the related art.
[Reference Example 4: evaluation of heat retention property of modified fibroin]
[0271] Dimethyl sulfoxide (DMSO) in which LiCl was dissolved at a concentration of 4.0 mass%
was prepared as a solvent, and the lyophilized powder of the modified fibroin obtained
in Test Example 2 was added thereto at a concentration of 24 mass%, and dissolved
using a shaker for 3 hours. Then, insoluble substances and bubbles were removed to
obtain a modified fibroin solution (spinning stock solution).
[0272] The prepared spinning stock solution was filtered at 60°C with a metal filter having
an opening of 5 µm, and then left in a 30 mL stainless syringe, bubbles were removed,
and then the solution was discharged from a solid nozzle having a needle diameter
of 0.2 mm to a 100 mass% methanol solidification tank. The discharge temperature was
60°C. After coagulation, the obtained raw yarn was wound and naturally dried to obtain
a modified fibroin fiber (raw fiber).
[0273] For comparison, regarding raw fibers, commercially available wool fibers, silk fibers,
cotton fibers, rayon fibers and polyester fibers were prepared.
[0274] Knitted fabrics were manufactured using the raw fibers according to flat knitting
using a flat knitting machine. The knitted fabric obtained using the PRT966 fibers
as raw fibers had a yarn number of 30 Nm, a number of twists of 1, a gauge number
of 18GG, and a basis weight of 90.1 g/m
2. The knitted fabric obtained using the PRT799 fibers as raw fibers had a yarn number
of 30 Nm, a number of twists of 1, a gauge number of GG: 16 and a basis weight of
111.0 g/m
2. The thickness and the gauge number of the knitted fabric obtained using other raw
fibers were adjusted such that the cover factor was almost the same as that of the
knitted fabric obtained using the PRT966 fiber and the PRT799 fibers. The details
were as follows.
Wool yarn number: 30 Nm, number of twists: 2, gauge number: 14GG, basis weight: 242.6
g/m2
Silk yarn number: 60 Nm, number of twists: 2, gauge number: 14GG, basis weight: 225.2
g/m2
Cotton yarn number: 34 Nm, number of twists: 2, gauge number: 14GG, basis weight:
194.1 g/m2
Rayon yarn number: 38 Nm, number of twists: 1, gauge number: 14GG, basis weight: 181.8
g/m2
Polyester yarn number: 60 Nm, number of twists: 1, gauge number: 14GG, basis weight:
184.7 g/m2
[0275] The heat retention property was evaluated using a KES-F7 Thermo Lab II tester (commercially
available from Kato Tech Co., Ltd.) and a dry contact method (a method assuming that
the skin and clothes were in direct contact with each other in a dry state). One piece
of knitted fabric cut into 20 cm×20 cm was prepared as a test piece (sample). The
test piece was set on a hot plate set to a certain temperature (30°C) and the amount
of heat (a) dissipated through the test piece was determined under conditions of a
wind velocity in a wind tunnel of 30 cm/sec. The amount of heat (b) dissipated was
determined under the same conditions as above without setting the test piece, and
the heat retention rate (%) was calculated according to the following formula.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0019)
[0276] The heat retention index was determined from the measurement results according to
the following Formula B.
![](https://data.epo.org/publication-server/image?imagePath=2021/14/DOC/EPNWA1/EP19782354NWA1/imgb0020)
[0277] Table 16 shows the calculation results of the heat retention index. A material having
a higher heat retention index can be evaluated as having a superior heat retention
property.
[Table 16]
Raw fiber |
Heat retention index |
PRT966 |
0.33 |
PRT799 |
0.22 |
Wool |
0.16 |
Silk |
0.11 |
Cotton |
0.13 |
Rayon |
0.02 |
Polyester |
0.18 |
[0278] As shown in Table 16, it was found that the modified fibroins (PRT966 and PRT799)
had a higher heat retention index and a superior heat retention property than materials
in the related art.
Reference Signs List