Technical Field
[0001] The present invention relates to a manufacturing method for a protein crimped staple
and particularly relates to a manufacturing method for a crimped staple of an artificial
fibroin including a modified fibroin.
Background Art
[0002] A protein fiber, unlike a synthetic fiber, has biodegradability and low energy consumption
for production and processing, and thus demands for various fields are expected to
increase in response to the recent increase in environmental consciousness.
[0003] As the natural protein fiber, a filament such as silk and a staple such as wool are
known. The former has a supple texture, and the latter has a soft feel and a heat
retaining property, each of which has own characteristics thereof.
[0004] Recently, attempts have been made to process a protein fiber and apply the protein
fiber to a wider range of applications. For example, in Patent Literatures 1 and 2,
a method for manufacturing a long-fiber nonwoven fabric or a long-fiber crimped yarn
by crimping the natural silk filament has been proposed. Further, the manufacturing
of a protein crimped staple from protein filaments and obtaining spun yarn, nonwoven
fabric, and the like using the protein crimped staple have also been studied in some
fields.
[0005] As a method for obtaining a protein crimped staple from protein filaments, for example,
a method of cutting a silk crimped filament crimped by the crimping processing method
disclosed in the above patent literatures can be considered.
Citation List
Patent Literature
[0006]
[Patent Literature 1] Japanese Unexamined Patent Publication No. 2006-207069
[Patent Literature 2] Japanese Unexamined Patent Publication No. H9-119033
Summary of Invention
Technical Problem
[0007] However, since in the crimping method described in Patent Literature 1, a protein
filament is crimped by a mechanical processing method such as an indentation method,
in a case where a protein crimped staple is manufactured using this crimping method,
there have been problems that a dedicated crimping device is required and the processing
cost is high. Further, in the crimping method described in Patent Literature 2, it
is necessary to perform a pretreatment for imparting a non-twisted latent crimping
property to the natural silk prior to the crimping process. Therefore, in a case where
this crimping method is used for manufacturing the protein crimped staple, there have
been problems that the number of processes is increased and the productivity is inevitably
decreased.
[0008] The present invention has been made in consideration of the above problems, and an
object of the present invention is to provide a method for efficiently manufacturing
a protein crimped staple from protein filaments at low cost.
Solution to Problem
[0009] The present invention relates to, for example, each of the following inventions.
- [1] A manufacturing method for a protein crimped staple, including:
- a) preparing an artificial fibroin filament containing a modified fibroin;
- b) cutting the artificial fibroin filament to obtain an artificial fibroin staple;
and
- c) performing crimping by bringing the artificial fibroin filament into contact with
an aqueous medium to crimp the artificial fibroin filament before the cutting or bringing
the artificial fibroin staple into contact with an aqueous medium to crimp the artificial
fibroin staple after the cutting.
- [2] The manufacturing method for a protein crimped staple [1], in which a shrinkage
rate after drying of the artificial fibroin filament defined by the following expression
is more than 7%.
- [3] The manufacturing method for a protein crimped staple according to [1] or [2],
in which a shrinkage rate when wetted of the artificial fibroin filament defined by
the following expression is 2% or more.
- [4] The manufacturing method for a protein crimped staple according to any one of
[1] to [3], in which the modified fibroin is a modified spider silk fibroin, and the
artificial fibroin filament is an artificial spider silk fibroin filament.
- [5] The manufacturing method for a protein crimped staple according to any one of
[1] to [4], in which the aqueous medium used in the performing crimping is a liquid
or gas containing water and having a temperature of 10°C to 230°C.
- [6] The manufacturing method for a protein crimped staple according to any one of
[1] to [5], in which the performing crimping further includes performing drying after
the bringing the artificial fibroin filament or the artificial fibroin staple into
contact with the aqueous medium.
- [7] The manufacturing method for a protein crimped staple according to any one of
[1] to [6], in which the aqueous medium used in the performing crimping contains a
volatile solvent.
Advantageous Effects of Invention
[0010] In the manufacturing method for a protein crimped staple of the present invention,
by adopting a simple and unique crimping process of simply bring a raw material protein
fiber into contact with an aqueous medium without using a dedicated crimping device,
a protein crimped staple can be easily and efficiently manufactured at low cost.
Brief Description of Drawings
[0011]
Fig. 1 is a schematic diagram illustrating one example of a domain sequence of a modified
fibroin.
Fig. 2 is a schematic diagram illustrating one example of a domain sequence of a modified
fibroin.
Fig. 3 is a schematic diagram illustrating one example of a domain sequence of a modified
fibroin.
Fig. 4 is an explanatory view schematically illustrating one example of a spinning
device for manufacturing a protein filament.
Fig. 5 is a photograph of a protein crimped staple obtained in Example 1.
Fig. 6 is another photograph of a protein crimped staple obtained in Example 1.
Fig. 7 is a photograph of a protein uncrimped staple obtained in Comparative Example.
Description of Embodiments
[0012] A manufacturing method for a protein crimped staple according to one aspect of the
present invention includes a process a, a process b, and a process c, each of which
will be described below, and the process b and the process c are in no particular
order. That is, the method may be performed in an order of the process a, the process
b, and the process c, or in an order of the process a, the process c, and the process
b.
<Process a>
[0013] The process is a process of preparing an artificial fibroin filament containing a
modified fibroin. Here, the filament (also referred to as "long fiber") and the staple
(also referred to as "short fiber") are obvious to those skilled in the art.
(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 (an N-terminal sequence and
a C-terminal sequence) may be further added to either or both of the N-terminal side
and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal
sequence, although not limited thereto, are typically regions that do not have repetitions
of amino acid motifs characteristic of fibroin and consist of amino acids of about
100 residues.
[0015] The term "modified fibroin" in the present specification means an artificially produced
fibroin (an artificial fibroin). The modified fibroin may be a fibroin in which the
domain sequence is different from the amino acid sequence of a fibroin derived from
the natural fibroin or may be the same as the amino acid sequence of a fibroin derived
from the natural fibroin. The "fibroin derived from the natural fibroin" referred
to in the present 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] As the "modified fibroin", an amino acid sequence of a fibroin derived from the natural
fibroin may be directly used, a fibroin whose amino acid sequence has been modified
based on an amino acid sequence of a fibroin derived from the natural fibroin (for
example, a fibroin whose amino acid sequence has been modified by altering a cloned
gene sequence of a fibroin derived from the natural fibroin) may be used, or a fibroin
artificially designed and synthesized independently of a fibroin derived from the
natural fibroin (for example, a fibroin having a desired amino acid sequence by chemically
synthesizing a nucleic acid encoding the designed amino acid sequence) may be used,
as long as it has the amino acid sequence specified in the present embodiment.
[0017] The term "domain sequence" in the present specification refers to an amino acid sequence
which produces a crystalline region (typically, corresponds to (A)
n motif of an amino acid sequence) and an amorphous region (typically, corresponds
to REP of an amino acid sequence) peculiar to fibroin and means 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 including alanine residues, and the
number of amino acid residues in the (A)
n motif is 2 to 27. The number of amino acid residues in the (A)
n motif may be 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. Further, the proportion 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 the (A)
n motif is composed of only alanine residues). In a plurality of (A)
n motifs present in the domain sequence, at least seven of the (A)
n motif may be composed of only alanine residues. REP represents an amino acid sequence
composed of 2 to 200 amino acid residues. The REP may represent an amino acid sequence
composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and
may be an integer of 10 to 300. The plurality of (A)
n motifs may have the same amino acid sequence or amino acid sequences different from
each other. The plurality of REPs may have the same amino acid sequence or amino acid
sequences different from each other.
[0018] The modified fibroin can be obtained, for example, by carrying out the modification
of an amino acid sequence equivalent to the substitution, deletion, insertion and/or
addition of one or a plurality of amino acid residues with respect to, for example,
a cloned gene sequence of a fibroin derived from the natural fibroin. The substitution,
deletion, insertion, and/or addition of an amino acid residue may be carried out by
methods well known to those skilled in the art, such as site-directed mutagenesis.
Specifically, the modifications may be carried out by methods described in literature
such as
Nucleic Acid Res. 10, 6487 (1982) and
Methods in Enzymology, 100, 448 (1983).
[0019] The fibroin derived from the natural 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 specifically, for example, a fibroin produced by insects or spiders.
[0020] Examples of the fibroin produced by insects include silk proteins produced by silkworms
such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna
pyretorum, Philosamia Cynthia ricini, Samia cynthia, Caligula japonica, Antheraea
mylitta, and Antheraea assama; and hornet silk proteins discharged by larvae of Vespa
simillima xanthoptera.
[0021] A more specific example of the fibroin produced by insects includes a silkworm fibroin
L chain (GenBank Accession No. M76430 (base sequence), AAA27840.1 (amino acid sequence)).
[0022] Examples of the fibroin produced by spiders include spider silk proteins produced
by spiders belonging to the genus Araneus such as Araneus ventricosus, Araneus diadematus,
Araneus pinguis, 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 Pronous minutes,
spiders belonging to the genus Cyrtarachne such as Cyrtarachne bufo and Cyrtarachne
inaequalis, spiders belonging to the genus Gasteracantha such as Gasteracantha kuhli
and Gasteracantha mammosa, 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 bruennich, 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 Cyrtophora such as Cyrtophora moluccensis,
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 magnifica, 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 Dyschiriognatha tenera, spiders belonging to
the genus Latrodectus such as Latrodectus mactans, Latrodectus hasseltii, Latrodectus
geometricus and Latrodectus tredecimguttatus, and spiders belonging to the family
Tetragnathidae such as spiders belonging to the genus Euprosthenops. Examples of spider
silk proteins include traction yarn proteins such as MaSp (MaSp1 and MaSp2) and ADF
(ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
[0023] More specific examples of the spider silk protein produced by spiders include fibroin-3
(adf-3) [derived from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid
sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus]
(GenBank Accession No. AAC47011 (amino acid sequence), U47856 (base sequence)), dragline
silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504
(amino acid sequence), U37520 (base sequence)), major ampullate spidroin 1 [derived
from Latrodectus hesperus] (GenBank Accession No. ABR68856 (amino acid sequence),
EF595246 (base sequence)), dragline silk protein spidroin 2 [derived from Nephila
clavata] (GenBank Accession No. AAL32472 (amino acid sequence), AF441245 (base sequence)),
major ampullate spidroin 1 [derived from Euprosthenops australis] (GenBank Accession
No. CAJ00428 (amino acid sequence), AJ973155 (base sequence)) and major ampullate
spidroin 2 [Euprosthenops australis] (GenBank Accession No. CAM32249.1 (amino acid
sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes]
(GenBank Accession No. AAC14589.1 (amino acid sequence), minor ampullate silk protein
2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), and
minor ampullate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No.
ABR37278.1 (amino acid sequence)).
[0024] As a further specified example of the fibroin derived from the natural fibroin, a
fibroin whose sequence information is registered in NCBI GenBank may be mentioned.
For example, sequences thereof may be confirmed by extracting sequences in which spidroin,
ampullate, fibroin, "silk and polypeptide", or "silk and protein" is described as
a keyword in DEFINITION among sequences containing INV as DIVISION in sequence information
registered in NCBI GenBank, sequences in which a specific character string of a product
is described from CDS, or sequences in which a specific character string is described
from SOURCE to TISSUE TYPE.
[0025] The modified fibroin may be a modified silk fibroin (a modified silk protein obtained
by modifying an amino acid sequence of a silk protein produced by silkworm), and a
modified spider silk fibroin (a modified spider silk protein obtained by modifying
an amino acid sequence of a spider silk protein produced by spiders). Among them,
a modified spider silk fibroin is preferably used.
[0026] The specific examples of the modified fibroin include a modified fibroin derived
from a large spinal canal thread protein produced in the major ampullate gland of
a spider, a modified fibroin having a reduced content of the glycine residue, a modified
fibroin having a reduced content of the (A)
n motif, and a modified fibroin having a reduced content of the glycine residue and
a reduced content of the (A)
n motif.
[0027] As the modified fibroin derived from a large spinal canal thread protein produced
in the major ampullate gland of a spider, a protein including a domain sequence represented
by Formula 1: [(A)
n motif - REP]
m is mentioned. In the modified fibroin derived from a large spinal canal thread protein
produced in the major ampullate gland of a spider, 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, even more preferably an integer of 10
to 20, even further 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 modified
fibroin derived from a large spinal canal thread protein produced in the major ampullate
gland of a spider, the number of amino acid residues constituting REP in Formula 1
is preferably 10 to 200 residues, more preferably 10 to 150 residues, and still more
preferably 20 to 100 residues, and even more preferably 20 to 75 residues. In the
modified fibroin derived from a large spinal canal thread protein produced in the
major ampullate gland of a spider, the number of residues in 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 modified fibroin derived from a large spinal canal thread protein produced in
the major ampullate gland of a spider may be a polypeptide including an amino acid
sequence unit represented by Formula 1: [(A)
n motif - REP]
m, and being an amino acid sequence in which the C-terminal sequence has the amino
acid sequence set forth in any of SEQ ID NOs: 14 to 16 or an amino acid sequence in
which the C-terminal sequence has an amino acid sequence having 90% or more homology
with the amino acid sequence set forth in any of SEQ ID NOs: 14 to 16.
[0029] The amino acid sequence set forth in SEQ ID NO: 14 is identical to the amino acid
sequence consisting of 50 amino acid residues at the C-terminal of the amino acid
sequence of ADF3 (GI: 1263287, NCBI). The amino acid sequence set forth in SEQ ID
NO: 15 is identical to the amino acid sequence obtained by removing 20 residues from
the C-terminal of the amino acid sequence set forth in SEQ ID NO: 14. The amino acid
sequence set forth in SEQ ID NO: 16 is identical to the amino acid sequence obtained
by removing 29 residues from the C-terminal of the amino acid sequence set forth in
SEQ ID NO: 14.
[0030] More specific examples of the modified fibroin derived from a large spinal canal
thread protein produced in the major ampullate gland of a spider include a modified
fibroin including (1-i) the amino acid sequence set forth in SEQ ID NO: 17 or (1-ii)
the amino acid sequence having 90% or more sequence identity with the amino acid sequence
set forth in SEQ ID NO: 17. The sequence identity is preferably 95% or more.
[0031] The amino acid sequence set forth in SEQ ID NO: 17 is an amino acid sequence obtained
by approximately doubling repeating regions from the first repeating region to the
13th repeating region and performing mutation so that translation is terminated at
the 1154th amino acid residue in an amino acid sequence obtained by adding the amino
acid sequence (SEQ ID NO: 18) consisting of a start codon, a His10 tag, and a recognition
site for HRV3C protease (human rhinovirus 3C protease) to the N-terminal of ADF3.
The C-terminal amino acid sequence of the amino acid sequence set forth in SEQ ID
NO: 17 is identical to the amino acid sequence set forth in SEQ ID NO: 16.
[0032] The modified fibroin of (1-i) may consist of the amino acid sequence set forth in
SEQ ID NO: 17.
[0033] The domain sequence of the modified fibroin having a reduced content of the glycine
residue has an amino acid sequence with a reduced content of the glycine residue,
as compared with a fibroin derived from the natural fibroin. It can be said that the
modified fibroin has an amino acid sequence equivalent to an amino acid sequence in
which at least one or a plurality of glycine residues in REP are substituted with
other amino acid residues, as compared with a fibroin derived from the natural fibroin.
[0034] The domain sequence of the modified fibroin having a reduced content of the glycine
residue may have an amino acid sequence equivalent to an amino acid sequence in which
one glycine residue in at least one or the plurality of motif sequences, at least
one of which is selected from 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, is substituted with other amino acid residue, as compared with a
fibroin derived from the natural fibroin.
[0035] In the modified fibroin having a reduced content of the glycine residue, the proportion
of the motif sequences in which the above-described glycine residue is substituted
with other amino acid residue may be 10% or more with respect to the entire motif
sequences.
[0036] The modified fibroin having a reduced content of the glycine residue may include
a domain sequence represented by Formula 1: [(A)
n motif - REP]
m and have an amino acid sequence in which z/w is 30% or more, 40% or more, 50% or
more, or 50.9% or more, in a case where the total number of amino acid residues consisting
of XGX (where G represents a glycine residue and X represents an amino acid residue
other than glycine) included in all REPs in the sequence excluding a sequence from
the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, is denoted by z, and the total number of amino acid residues
in the amino acid sequence excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence, is denoted by w. The proportion of 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 even still more preferably 100% (which means that
the (A)
n motif consists of only alanine residues).
[0037] In the modified fibroin having a reduced content of the glycine residue, the content
proportion of an amino acid sequence consisting of XGX is preferably increased by
substituting one glycine residue in GGX motif with other amino acid residue. In the
modified fibroin having a reduced content of the glycine residue, the content proportion
of an amino acid sequence consisting of GGX in the domain sequence is preferably 30%
or less, more preferably 20% or less, still more preferably 10% or less, even still
more preferably 6% or less, still further preferably 4% or less, and particularly
preferably 2% or less. The content proportion of an amino acid sequence consisting
of GGX in a domain sequence can be calculated by the same method as the following
method for calculating the content proportion (z/w) of the amino acid sequence consisting
of XGX.
[0038] The calculation method of z/w will be described in more detail. First, in a fibroin
(a modified fibroin or a fibroin derived from the natural fibroin) including a domain
sequence represented by Formula 1: ([(A)
n motif - REP]
m], the amino acid sequence consisting of XGX is extracted from all REPs included in
a sequence excluding a sequence from the (A)
n motif located 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, in a case where 50 amino acid sequences consisting of XGX (without
overlap) are extracted, z is 50 × 3 = 150. Further, for example, in a case where there
exists an X (a central X) contained in two XGXs, as in the case of an amino acid sequence
consisting of XGXGX, the calculation is performed by subtracting the overlapping portion
(in the case of XGXGX, it is counted as 5 amino acid residues). w is the total number
of amino acid residues included in the sequence excluding a sequence from the (A)
n motif located 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 illustrated
in Fig. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (the (A)
n motif located closest to the C-terminal side is excluded.). Next, z/w (%) can be
calculated by dividing z by w.
[0039] In the modified fibroin having a reduced content of the glycine residue, z/w is preferably
50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more,
even still more preferably 70% or more, and still further preferably 80% or more.
The upper limit of z/w is not particularly limited, but, for example, it may be 95%
or less.
[0040] The modified fibroin having a reduced content of the glycine residue cab be obtained
by, for example, modifying a cloned fibroin derived from the natural fibroin gene
sequence so that at least a part of a base sequence encoding a glycine residue is
substituted with other amino acid residue to encode other amino acid residue. In this
case, one glycine residue in GGX motif and GPGXX motif may be selected as the glycine
residue to be modified or may be substituted so that z/w is 50.9% or more. Alternatively,
a modified fibroin may also be obtained, for example, by designing an amino acid sequence
satisfying the above-described aspect based on the amino acid sequence of a fibroin
derived from the natural fibroin and chemically synthesizing a nucleic acid encoding
the designed amino acid sequence. In any case, with respect to the amino acid sequence
of a fibroin derived from the natural fibroin, in addition to the modification corresponding
to the substitution of the glycine residue in REP with other amino acid residue, further
modification of amino acid sequence corresponding to substitution, deletion, insertion
and/or addition of one or a plurality of amino acid residues may be carried out.
[0041] The other amino acid residue described above is not particularly limited as long
as it is an amino acid residue other than glycine residue, but it is preferably a
hydrophobic amino acid residue such as valine (V) residue, leucine (L) residue, isoleucine
(I) residue, methionine (M) residue, proline (P) residue, phenylalanine (F) residue,
and tryptophan (W) residue, or a hydrophilic amino acid residues such glutamine (Q)
residue, asparagine (N) residue, serine (S) residue, lysine (K) residue, and glutamic
acid (E) residue, more preferably valine (V) residue, leucine (L) residue, isoleucine
(I) residue, and glutamine (Q) residue, and still more preferably glutamine (Q) residue.
[0042] A more specific example of the modified fibroin having a reduced content of the glycine
residues includes a modified fibroin including an amino acid sequence having (2-i)
the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or
SEQ ID NO: 12, or (2-ii) an amino acid sequence having 90% or more sequence identity
with the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10,
or SEQ ID NO: 12.
[0043] The modified fibroin of (2-i) will be described. The amino acid sequence set forth
in SEQ ID NO: 3 is obtained by substituting all GGXs in REP of the amino acid sequence
set forth in SEQ ID NO: 1 equivalent to a fibroin derived from the natural fibroin
with GQX. The amino acid sequence set forth in SEQ ID NO: 4 is obtained by deleting
one of every two (A)
n motifs from the N-terminal side to the C-terminal side in the amino acid sequence
set forth in SEQ ID NO: 3 and further inserting one [(A)
n motif - REP] just before the C-terminal sequence. The amino acid sequence set forth
in SEQ ID NO: 10 is obtained by inserting two alanine residues at the C-terminal side
of each (A)
n motif of the amino acid sequence set forth in SEQ ID NO: 4, and further substituting
a part of glutamine (Q) residues with serine (S) residues and deleting a part of amino
acids on the N-terminal side so that the molecular weight thereof is approximately
the same as that of SEQ ID NO: 4. The amino acid sequence set forth in SEQ ID NO:
12 is an amino acid sequence obtained by adding a His tag to the C-terminal of a sequence
obtained by repeating, four times, a region of 20 domain sequences (where several
amino acid residues on the C-terminal side of the region are substituted) present
in the amino acid sequence set forth in SEQ ID NO: 9.
[0044] The value of z/w in the amino acid sequence set forth SEQ ID NO: 1 (corresponds to
a fibroin derived from the natural fibroin) is 46.8%. The values of z/w in the amino
acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ
ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 10, and the amino acid sequence
set forth in SEQ ID NO: 12 are respectively 58.7%, 70.1%, 66.1%, and 70.0%. In addition,
the values of x/y with a Giza ratio (described later) of 1:1.8 to 11.3 in the amino
acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10,
and SEQ ID NO: 12 are respectively 15.0%, 15.0%, 93.4%, 92.7%, and 89.3%.
[0045] The modified fibroin of (2-i) may consist of the amino acid sequence set forth in
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.
[0046] The modified fibroin of (2-ii) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 10, or SEQ ID NO: 12. The modified fibroin of (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.
[0047] The modified fibroin of (2-ii) preferably has 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or
SEQ ID NO: 12, and in a case where the total number of amino acid residues in the
amino acid sequence consisting of XGX (where G represents a glycine residue and X
represents an amino acid residue other than glycine) included in REP is z, and the
total number of amino acid residues in REP in the domain sequence is w, z/w is preferably
50.9% or more.
[0048] The above-described modified fibroin may include a tag sequence at either or both
of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect,
and visualize the modified fibroin.
[0049] The tag sequence may be, for example, an affinity tag utilizing specific affinity
(binding property, affinity) with another molecule. As a specific example of the affinity
tag, a histidine tag (a His tag) can be mentioned. The His tag is a short peptide
in which about 4 to 10 histidine residues are arranged and has a property of specifically
binding to a metal ion such as nickel, and thus it can be used for isolation of a
modified fibroin by a chelating metal chromatography. A specific example of the tag
sequence may be an amino acid sequence set forth in SEQ ID NO: 5 (an amino acid sequence
including a His tag).
[0050] In addition, a tag sequence such as glutathione-S-transferase (GST) that specifically
binds to glutathione or a maltose binding protein (MBP) that specifically binds to
maltose can also be used.
[0051] Further, an "epitope tag" utilizing an antigen-antibody reaction can also be used.
By adding a peptide (an epitope) showing antigenicity as a tag sequence, an antibody
against the epitope can be bound. Examples of the epitope tag include an HA (peptide
sequence of hemagglutinin of influenza virus) tag, a myc tag, and a FLAG tag. The
modified fibroin can be easily purified with high specificity by utilizing an epitope
tag.
[0052] It is also possible to use a tag sequence which can be cleaved with a specific protease.
By treating a protein adsorbed via the tag sequence with a protease, it is also possible
to recover a modified fibroin cleaved from the tag sequence.
[0053] A more specific example of the modified fibroin including a tag sequence may be a
modified fibroin including (2-iii) the amino acid sequence set forth in SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or (2-iv) an amino acid sequence
having 90% or more sequence identity with the amino acid sequence set forth in SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
[0054] The amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 are respectively amino acid sequences obtained
by adding the amino acid sequence (including a His tag and a hinge sequence) set forth
in SEQ ID NO: 5 to the N-terminal of the amino acid sequences set forth in SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12.
[0055] The modified fibroin of (2-iii) may consist of the amino acid sequence set forth
in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
[0056] The modified fibroin of (2-iv) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 11, or SEQ ID NO: 13. The modified fibroin of (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.
[0057] The modified fibroin of (2-iv) preferably has 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, or
SEQ ID NO: 13, and in a case where the total number of amino acid residues in the
amino acid sequence consisting of XGX (where G represents a glycine residue and X
represents an amino acid residue other than glycine) included in REP is z, and the
total number of amino acid residues in REP in the domain sequence is w, z/w is preferably
50.9% or more.
[0058] The above-mentioned modified fibroin may include a secretory signal for releasing
the protein produced in the recombinant protein production system to the outside of
a host. The sequence of the secretory signal can be appropriately set depending on
the type of the host.
[0059] The domain sequence of a modified fibroin having a reduced content of the (A)
n motif has an amino acid sequence with a reduced content of the (A)
n motif, as compared with a fibroin derived from the natural fibroin. It can be said
that the domain sequence of the modified fibroin has an amino acid sequence equivalent
to an amino acid sequence in which at least one or a plurality of (A)
n motifs are deleted, as compared with a fibroin derived from the natural fibroin.
[0060] The modified fibroin having a reduced content of the (A)
n motif may have an amino acid sequence equivalent to an amino acid sequence in which
10% to 40% of the (A)
n motifs are deleted from a fibroin derived from the natural fibroin.
[0061] The domain sequence of the modified fibroin having a reduced content of the (A)
n motif may have an amino acid sequence equivalent to an amino acid sequence obtained
by deleting one of every one to three (A)
n motifs from the N-terminal side to the C-terminal side, as compared with a fibroin
derived from the natural fibroin.
[0062] The domain sequence of the modified fibroin having a reduced content of the (A)
n motif may have an amino acid sequence equivalent to an amino acid sequence obtained
by repeating deletion of at least two consecutive (A)
n motifs and deletion of one (A)
n motif in this order from the N-terminal side to the C-terminal side, as compared
with a fibroin derived from the natural fibroin.
[0063] The domain sequence of the modified fibroin having a reduced content of the (A)
n motif may have an amino acid sequence equivalent to an amino acid sequence obtained
by deleting one of every two (A)
n motifs from the N-terminal side to the C-terminal side.
[0064] The modified fibroin having a reduced content of the (A)
n motif may include a domain sequence represented by Formula 1: [(A)
n motif - REP]
m, and in a case where the number of amino acid residues of two [(A)
n motif - REP] units adjacent to each other is sequentially compared from the N-terminal
side to the C-terminal side and then the number of amino acid residues of one REP
having a small number of amino acid residues is set to 1, the maximum total value
of the number of amino acid residues of two [(A)
n motif - REP] units adjacent to each other, in which the ratio of the number of amino
acid residues of the other REP is 1.8 to 11.3, is denoted by x, and the total number
of amino acid residues in the domain sequence is denoted by y, the modified fibroin
may have an amino acid sequence in which x/y is 20% or more, 30% or more, 40% or more,
or 50% or more. The proportion of 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 even still more preferably 100% (which means that
the (A)
n motif consists of only alanine residues).
[0065] The method for calculating x/y will be described in more detail with reference to
Fig. 1. Fig. 1 illustrates a domain sequence obtained by removing an N-terminal sequence
and a C-terminal sequence from a modified fibroin. The domain sequence has a sequence
of, from the N-terminal side (left side), (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 sequence.
[0066] Two [(A)
n motif - REP] units adjacent to each other are sequentially selected from the N-terminal
side toward the C-terminal side so that the units are not overlapped with each other.
In this case, an unselected [(A)
n motif - REP] unit may be present. In Fig. 1, pattern 1 (comparison of first REP and
second REP, and comparison of third REP and fourth REP), pattern 2 (comparison of
first REP and second REP, and comparison of fourth REP and fifth REP), pattern 3 (comparison
of second REP and third REP, and comparison of fourth REP and fifth REP), and pattern
4 (comparison of first REP and second REP). There are other selection methods other
than these methods.
[0067] Subsequently, for each pattern, the number of amino acid residues of each REP in
two selected [(A)
n motif - REP] units adjacent to each other is compared. The comparison is performed
by determining the ratio of the number of amino acid residues of one REP to the number
of amino acid residues of the other REP having the smaller number of amino acid residues
so that the number of amino acid residues in the other REP is set to 1. For example,
in the case of comparing the first REP (50 amino acid residues) and the second REP
(100 amino acid residues), when the first REP having the smaller number of amino acid
residues is set to 1, the ratio of the number of amino acid residues of the second
REP is 100/50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid
residues) and the fifth REP (30 amino acid residues), when the fourth REP having the
smaller number of amino acid residues is set to 1, the ratio of the number of amino
acid residues of the fifth REP is 30/20=1.5.
[0068] In Fig. 1, in a case where one group of [(A)
n motif - REP] units having the smaller number of amino acid residues is set to 1,
the other group in which the ratio of the number of amino acid residues is 1.8 to
11.3 is indicated by a solid line. Hereinafter, this ratio is referred to as a Giza
ratio. In a case where one group of [(A)
n motif - REP] units having the smaller number of amino acid residues is set to 1,
the other group in which the ratio of the number of amino acid residues is less than
1.8 or more than 11.3 is indicated by a broken line.
[0069] In each pattern, the total numbers of amino acid residues of two [(A)
n motif - REP] units adjacent to each other indicated by solid lines are added (not
only the number of REPs but also the number of the amino acid residues in the (A)
n motif are added.) Then, the added total values are compared, and the total value
(maximum value of the total values) of the pattern having the maximum total value
is denoted by x. In the example illustrated in Fig. 1, the total value of the pattern
1 is the maximum.
[0070] Next, x/y (%) can be calculated by dividing x by y which is the total number of the
amino acid residues of the domain sequence.
[0071] In the modified fibroin having a reduced content of the (A)
n motif, x/y is preferably 50% or more, more preferably 60% or more, still more preferably
65% or more, even still more preferably 70% or more, still further preferably 75%
or more, and particularly preferably 80% or more. The upper limit of x/y is not particularly
limited, but for example, it may be 100% or less. In a case where the Giza ratio is
1:1.9 to 11.3, x/y is preferably 89.6% or more. In a case where the Giza ratio is
1:1.8 to 3.4, x/y is more preferably 77.1% or more. In a case where the Giza ratio
is 1:1.9 to 8.4, x/y is still more preferably 75.9% or more. In a case where the Giza
ratio is 1:1.9 to 4.1, x/y is even still more preferably 64.2% or more.
[0072] In a case where the modified fibroin having a reduced content of the (A)
n motif is a modified fibroin in which at least seven (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, even still more preferably 60% or more, still further preferably 70% or more,
and particularly preferably 80% or more. The upper limit of x/y is not particularly
limited as long as it is 100% or less.
[0073] The modified fibroin having a reduced content of the (A)
n motif can be obtained, for example, by deleting one or a plurality sequences encoding
(A)
n motif from a cloned gene sequence of a fibroin derived from the natural fibroin so
that x/y is 64.2% or more. Alternatively, the modified fibroin having a reduced content
of the (A)
n motif may also be obtained, for example, by designing an amino acid sequence equivalent
to an amino acid sequence obtained by deleting one or a plurality (A)
n motifs so that x/y is 64.2% or more based on the amino acid sequence of a fibroin
derived from the natural fibroin and chemically synthesizing a nucleic acid encoding
the designed amino acid sequence. In any case, with respect to the amino acid sequence
of a fibroin derived from the natural fibroin, in addition to the modification corresponding
to the deletion of the (A)
n motif, further modification of amino acid sequence equivalent to substitution, deletion,
insertion and/or addition of one or a plurality of amino acid residues may be carried
out.
[0074] A more specific example of the modified fibroin having a reduced content of the (A)
n motif includes a modified fibroin including an amino acid sequence having (3-i) the
amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ
ID NO: 12, or (3-ii) an amino acid sequence having 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or
SEQ ID NO: 12.
[0075] The modified fibroin of (3-i) will be described. The amino acid sequence set forth
in SEQ ID NO: 2 is obtained by deleting one of every two (A)
n motifs from the N-terminal side to the C-terminal side in the amino acid sequence
set forth in SEQ ID NO: 1 equivalent to a fibroin derived from the natural fibroin
and by further inserting one [(A)
n motif - REP] just before the C-terminal sequence. The amino acid sequence set forth
in SEQ ID NO: 4 is obtained by substituting all GGXs in REP of the amino acid sequence
set forth in SEQ ID NO: 2 with GQX. The amino acid sequence set forth in SEQ ID NO:
10 is obtained by inserting two alanine residues at the C-terminal side of each (A)
n motif of the amino acid sequence set forth in SEQ ID NO: 4, and further substituting
a part of glutamine (Q) residues with serine (S) residues and deleting a part of amino
acids on the N-terminal side so that the molecular weight thereof is approximately
the same as that of SEQ ID NO: 4. The amino acid sequence set forth in SEQ ID NO:
12 is an amino acid sequence obtained by adding a His tag to the C-terminal of a sequence
obtained by repeating, four times, a region of 20 domain sequences (where several
amino acid residues on the C-terminal side of the region are substituted) present
in the amino acid sequence set forth in SEQ ID NO: 9.
[0076] The value of x/y with a Giza ratio of 1:1.8 to 11.3 in the amino acid sequence set
forth in SEQ ID NO: 1 (equivalent to a fibroin derived from the natural fibroin) is
15.0%. Both the values of x/y in the amino acid sequence set forth in SEQ ID NO: 2
and the value of x/y in the amino acid sequence set forth in SEQ ID NO: 4 are 93.4%.
The value of x/y in the amino acid sequence set forth in SEQ ID NO: 10 is 92.7%. The
value of x/y in the amino acid sequence set forth in SEQ ID NO: 12 is 89.3%. The values
of z/w in the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12 are respectively 46.8%, 56.2%, 70.1%, 66.1%,
and 70.0%.
[0077] The modified fibroin of (3-i) may consist of the amino acid sequence set forth in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.
[0078] The modified fibroin of (3-ii) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 10, or SEQ ID NO: 12. The modified fibroin of (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.
[0079] The modified fibroin of (3-ii) preferably has 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, or
SEQ ID NO: 12, and in a case where the number of amino acid residues of two [(A)
n motif - REP] units adjacent to each other is sequentially compared from the N-terminal
side to the C-terminal side, then the number of amino acid residues of one REP having
a small number of amino acid residues is set to 1, and the maximum total value of
the added numbers of amino acid residues of two [(A)
n motif - REP] units adjacent to each other, in which the ratio (1:1.8 to 11.3 as a
Giza ratio) of the number of amino acid residues of the other REP is 1.8 to 11.3,
is denoted by x, and the total number of amino acid residues in the domain sequence
is denoted by y, x/y is preferably 64.2% or more.
[0080] The modified fibroin described above may include a tag sequence described above at
either or both of the N-terminal and C-terminal.
[0081] A more specific example of the modified fibroin including a tag sequence may be a
modified fibroin including (3-iii) the amino acid sequence set forth in SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or (2-iv) an amino acid sequence
having 90% or more sequence identity with the amino acid sequence set forth in SEQ
ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
[0082] The amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 are respectively amino acid sequences obtained
by adding the amino acid sequence (including a His tag) set forth in SEQ ID NO: 5
to the N-terminal of the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 12.
[0083] The modified fibroin of (3-iii) may consist of the amino acid sequence set forth
in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.
[0084] The modified fibroin of (3-iv) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, or SEQ ID NO: 13. The modified fibroin of (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.
[0085] The modified fibroin of (3-iv) preferably has 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or
SEQ ID NO: 13, and in a case where the number of amino acid residues of two [(A)
n motif - REP] units adjacent to each other is sequentially compared from the N-terminal
side to the C-terminal side, then the number of amino acid residues of one REP having
a small number of amino acid residues is set to 1, the maximum total value of the
added numbers of amino acid residues of two [(A)
n motif - REP] units adjacent to each other, in which the ratio of the number of amino
acid residues of the other REP is 1.8 to 11.3, is denoted by x, and the total number
of amino acid residues in the domain sequence is denoted by y, x/y is preferably 64.2%
or more.
[0086] The above-mentioned modified fibroin may include a secretory signal for releasing
the protein produced in the recombinant protein production system to the outside of
a host. The sequence of the secretory signal can be appropriately set depending on
the type of the host.
[0087] The domain sequence of the modified fibroin having a reduced content of the glycine
residue and (A)
n motif has an amino acid sequence having not only a reduced content of the (A)
n motif but also having a reduced content of the glycine residue, as compared with
a fibroin derived from the natural fibroin. It can be said that the domain sequence
of the modified fibroin has an amino acid sequence equivalent to an amino acid sequence
in which at least one or a plurality of (A)
n motifs are deleted and further at least one or a plurality of glycine residues in
REP are substituted with other amino acid residues, as compared with a fibroin derived
from the natural fibroin. That is, the modified fibroin is a modified fibroin having
both of the characteristics of a modified fibroin having a reduced content of the
glycine residue and the characteristics of a modified fibroin having a reduced content
of the (A)
n motif. Specific aspects and the like are as described for a modified fibroin having
a reduced content of the glycine residue and a modified fibroin having a reduced content
of the (A)
n motif.
[0088] A more specific example of the modified fibroin having a reduced content of the glycine
residue and (A)
n motif includes a modified fibroin including an amino acid sequence having (4-i) the
amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, or
(4-ii) an amino acid sequence having 90% or more sequence identity with the amino
acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12. Specific
aspects of the modified fibroin including the amino acid sequence set forth in SEQ
ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12 are as described above.
[0089] A domain sequence of a modified fibroin according to another embodiment may have
an amino acid sequence locally containing a region with a high hydropathy index equivalent
to an amino acid sequence in which one or a plurality of amino acid residues in REP
are substituted with amino acid residues with a high hydropathy index and/or one or
a plurality of amino acid residues with a high hydropathy index are inserted into
REP, as compared with a fibroin derived from the natural fibroin.
[0090] It is preferable that the region locally having high hydropathy index is composed
of two to four consecutive amino acid residues.
[0091] It is more preferable that the amino acid residues with a high hydropathy index are
selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine
(C), methionine (M), and alanine (A).
[0092] The modified fibroin according to the present embodiment may further include an amino
acid sequence equivalent to an amino acid sequence in which one or a plurality of
amino acid residues are substituted, deleted, inserted and/or added, as compared with
a fibroin derived from the natural fibroin, in addition to the modification of the
amino acid sequence in which one or a plurality of amino acid residues in REP are
substituted with amino acid residues with a high hydropathy index and/or one or a
plurality of amino acid residues with a high hydropathy index are inserted into REP,
as compared with a fibroin derived from the natural fibroin of the present embodiment.
[0093] The modified fibroin according to the present embodiment may be obtained by, with
respect to a cloned gene sequence of a fibroin derived from the natural fibroin, substituting
one or a plurality of hydrophilic amino acid residues in REP (for example, amino acid
residues having a negative hydropathy index) with a hydrophobic amino acid residue
(for example, amino acid residues having a positive hydropathy index), and/or inserting
one or a plurality of hydrophobic amino acid residues into REP. Further, for example,
the modified fibroin may also be obtained by, for example, designing an amino acid
sequence equivalent to an amino acid sequence in which with respect to the amino acid
sequence of a fibroin derived from the natural fibroin, one or a plurality of hydrophilic
amino acid residues in REP are substituted with hydrophobic amino acid residues and/or
one or a plurality of hydrophobic amino acid residues are inserted into REP, and chemically
synthesizing a nucleic acid encoding the designed amino acid sequence. In any case,
with respect to the amino acid sequence a fibroin derived from the natural fibroin,
in addition to the modification corresponding to the substitution of one or a plurality
of hydrophilic amino acid residues in REP with hydrophobic amino acid residues and/or
insertion of one or a plurality of hydrophobic amino acid residues into REP, further
modification of amino acid sequence equivalent to substitution, deletion, insertion
and/or addition of one or a plurality of amino acid residues may be carried out.
[0094] Further, a modified fibroin according to another embodiment may include a domain
sequence represented by Formula 1: [(A)
n motif - REP]
m and have an amino acid sequence in which p/q is 6.2% or more, in a case where in
all REPs included in a sequence excluding a sequence from an (A)
n motif located 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 contained in a region
where an average value of hydropathy indices of the four consecutive amino acid residues
is 2.6 or more is denoted by p, and the total number of amino acid residues contained
in the sequence excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is denoted by q.
[0095] Regarding the hydropathy index of amino acid residues, known indices from (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 as a reference. Specifically, the hydropathy index (hereinafter, also
referred to as "HI") of each amino acid is as shown in Table 1 below.
[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 |
[0096] The calculation method of p/q will be described in more detail. In the calculation,
the sequence (hereinafter, also referred to as "sequence A") excluding a sequence
from the (A)
n motif located 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, average values of hydropathy
indices of the four consecutive amino acid residues are calculated. The average value
of the hydropathy indices is obtained by dividing the total sum of HI of each of the
amino acid residues contained in the four consecutive amino acid residues by 4 (the
number of amino acid residues). The average value of the hydropathy indices is obtained
for all of the four consecutive amino acid residues (each of the amino acid residues
is used for calculating the average value 1 to 4 times). Next, a region where the
average value of the hydropathy indices of the four consecutive amino acid residues
is 2.6 or more is specified. Even in a case where a plurality of certain amino acid
residues correspond to the "four consecutive amino acid residues having an average
value of the hydropathy indices of 2.6 or more", the amino acid residue is counted
as one amino acid residue in the region. The total number of amino acid residues included
in the region is denoted by p. The total number of amino acid residues included in
the sequence A is denoted by q.
[0097] For example, in a case where the "four consecutive amino acid residues whose average
value of the hydropathy indices is 2.6 or more" are extracted from 20 places (without
overlap), in the region where the average value of the hydropathy indices of the four
consecutive amino acid residues is 2.6 or more, the number of the four consecutive
amino acid residues (without overlap) is 20, and thus p is 20 × 4 = 80. In addition,
for example, in a case where two of the "four consecutive amino acid residues having
an average value of the hydropathy indices of 2.6 or more" overlap by only one amino
acid residue, in the region where the average value of the hydropathy indices of the
four consecutive amino acid residues is 2.6 or more, the number of amino acid residues
being included is 7 (p = 2 × 4 - 1 = 7. "-1" corresponds to the subtraction of the
overlapping portion). For example, in the case of the domain sequence shown in Fig.
2, since the number of the "four consecutive amino acid residues having an average
value of the hydropathy indices of 2.6 or more", which do not overlap, is 7, p is
7 × 4 = 28. Further, for example, in the case of the domain sequence illustrated in
Fig. 2, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (the (A)
n motif present closest to the C-terminal side can not be included). Next, p/q (%)
can be calculated by dividing p by q. In the case of Fig. 2, p/q (%) is 28/170 = 16.47%.
[0098] In the modified fibroin according to the present embodiment, p/q is preferably 6.2%
or more, more preferably 7% or more, still more preferably 10% or more, even still
more preferably 20% or more, and still further preferably 30% or more. The upper limit
of p/q is not particularly limited, but for example, it may be 45% or less.
[0099] The modified fibroin according to the present embodiment may be obtained by, for
example, modifying an amino acid sequence of a cloned a fibroin derived from the natural
fibroin to an amino acid sequence locally containing a region locally having a high
hydropathy index by substituting one or a plurality of hydrophilic amino acid residues
in REP (for example, amino acid residues having a negative hydropathy index) with
hydrophobic amino acid residues (for example, amino acid residues having a positive
hydropathy index), and/or inserting one or a plurality of hydrophobic amino acid residues
into REP, so that the p/q condition is satisfied. Alternatively, the modified fibroin
may also be obtained, for example, by designing an amino acid sequence satisfying
the p/q condition based on the amino acid sequence of a fibroin derived from the natural
fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid
sequence. In any case, in addition to the modification corresponding to the substitution
of one or a plurality of amino acid residues in REP with amino acid residues with
a high hydropathy index and/or insertion of one or a plurality of amino acid residues
with a high hydropathy index into REP, as compared with the amino acid sequence of
a fibroin derived from the natural fibroin, further modification corresponding to
substitution, deletion, insertion, and/or addition of one or a plurality of amino
acid residues may be carried out.
[0100] The amino acid residue with a high hydropathy index is preferably isoleucine (I),
valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine
(A), and more preferably valine (V), leucine (L), and isoleucine (I), but is not particularly
limited thereto.
[0101] Another specific example of the modified fibroin may be a modified fibroin including
(5-iii) the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ
ID NO: 23, or (5-ii) an amino acid sequence having 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.
[0102] The modified fibroin of (5-i) will be described. The amino acid sequence set forth
in SEQ ID NO: 19 is obtained by deleting a part of the amino acid sequence of the
consecutive alanine residues in the (A)
n motif of a fibroin derived from the natural fibroin so that the number of the consecutive
alanine residues in the (A)
n motif is five. The amino acid sequence set forth in SEQ ID NO: 20 is obtained by
inserting an amino acid sequence consisting of three amino acid residues (VLI) at
two sites for each REP with respect to the amino acid sequence set forth in SEQ ID
NO: 19, and deleting a part of the amino acids on the C-terminal side therefrom so
that the molecular weight thereof is approximately the same as that of the amino acid
sequence set forth in SEQ ID NO: 19. The amino acid sequence set forth in SEQ ID NO:
21 is obtained by inserting two alanine residues at the C-terminal side of each (A)
n motif with respect to the amino acid sequence set forth in SEQ ID NO: 19, and further
substituting a part of glutamine (Q) residues with serine (S) residues and deleting
a part of amino acids on the C-terminal side so that the molecular weight thereof
is approximately the same as that of the amino acid sequence set forth in SEQ ID NO:
19. The amino acid sequence set forth in SEQ ID NO: 22 is obtained by inserting an
amino acid sequence consisting of three amino acid residues (VLI) at one site for
each REP with respect to the amino acid sequence set forth in SEQ ID NO: 21. The amino
acid sequence set forth in SEQ ID NO: 23 is obtained by inserting an amino acid sequence
consisting of three amino acid residues (VLI) at two sites for each REP with respect
to the amino acid sequence set forth in SEQ ID NO: 21.
[0103] The modified fibroin of (5-i) may consist of the amino acid sequence set forth in
SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.
[0104] The modified fibroin of (5-ii) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID
NO: 22, or SEQ ID NO: 23. The modified fibroin of (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.
[0105] The modified fibroin of (5-ii) preferably has 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23,
and preferably has an amino acid sequence in which p/q is 6.2% or more, in a case
where in all REPs included in a sequence excluding a sequence from the (A)
n motif located 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 contained in a region
where an average value of hydropathy indices of the four consecutive amino acid residues
is 2.6 or more is denoted by p, and the total number of amino acid residues contained
in the sequence excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is denoted by q.
[0106] The above-described modified fibroin may include a tag sequence at either or both
of the N-terminal and C-terminal.
[0107] A more specific example of the modified fibroin including a tag sequence may be a
modified fibroin including (5-iii) the amino acid sequence set forth in SEQ ID NO:
24, SEQ ID NO: 25, or SEQ ID NO: 26, or (5-iv) an amino acid sequence having 90% or
more sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ
ID NO: 25, or SEQ ID NO: 26.
[0108] The amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO:
26 are respectively amino acid sequences obtained by adding the amino acid sequence
(including a His tag and a hinge sequence) set forth in SEQ ID NO: 5 to the N-terminal
of the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID
NO: 23.
[0109] The modified fibroin of (5-iii) may consist of the amino acid sequence set forth
in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
[0110] The modified fibroin of (5-iv) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID
NO: 25, or SEQ ID NO: 26. The modified fibroin of (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.
[0111] The modified fibroin of (5-iv) preferably has 90% or more sequence identity with
the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26,
and preferably has an amino acid sequence in which p/q is 6.2% or more, in a case
where in all REPs included in a sequence excluding a sequence from the (A)
n motif located 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 contained in a region
where an average value of hydropathy indices of the four consecutive amino acid residues
is 2.6 or more is denoted by p, and the total number of amino acid residues contained
in the sequence excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is denoted by q.
[0112] The above-mentioned modified fibroin may include a secretory signal for releasing
the protein produced in the recombinant protein production system to the outside of
a host. The sequence of the secretory signal can be appropriately set depending on
the type of the host.
[0113] A modified fibroin according to another embodiment has an amino acid sequence with
a reduced content of the glutamine residue, as compared with a fibroin derived from
the natural fibroin.
[0114] It is preferable that the modified fibroin according to the present embodiment includes
at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence
of REP.
[0115] In a case where the modified fibroin according to the present embodiment includes
a GPGXX motif in REP, a GPGXX motif content rate is usually 1% or more, may be 5%
or more, and is preferably 10% or more. The upper limit of the GPGXX motif content
rate is not particularly limited, may be 50% or less, and may be 30% or less.
[0116] In the present specification, the "GPGXX motif content rate" is a value calculated
by the following method.
[0117] In a fibroin (a fibroin derived from the natural 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 a case where the number obtained by tripling the total number of the GPGXX
motifs included in all REPs included in a sequence excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence (that is, equivalent to the total number of G and P in the
GPGXX motifs) is denoted by s, and the total number of amino acid residues in all
REPs excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence and further excluding (A)
n motifs is denoted by t, the GPGXX motif content rate is calculated as s/t.
[0118] For the calculation of the GPGXX motif content rate, the "sequence excluding a sequence
from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" is used to exclude the effect occurring due to the fact
that the "sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal from the domain sequence"
(sequence equivalent to REP) may include a sequence that is not correlated with the
sequence characteristics of fibroin, which influences the calculation result of the
GPGXX motif content rate in a case where m is small (that is, in case a where the
domain sequence is short). In a case where a "GPGXX motif" is located at the C-terminal
of REP, it is treated as "GPGXX motif" even in a case where "XX" is, for example,
"AA".
[0119] Fig. 3 is a schematic diagram showing a domain sequence of a modified fibroin. The
calculation method of the GPGXX motif content rate will be specifically described
with reference to Fig. 3. First, in a domain sequence of a modified fibroin (which
is an [(A)
n motif - REP]
m - (A)
n motif] type) illustrated in Fig. 3, since all REPs are included in the "sequence
excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" (in Fig. 3, shown as "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 excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" (in Fig. 1, shown as "region A"), t which is the total number
of amino acid residues in all REPs excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence and further excluding (A)
n motifs, is 50 + 40 + 10 + 20 + 30 = 150. Next, s/t (%) can be calculated by dividing
s by t and is 21/150 = 14.0% in the case of the modified fibroin of Fig. 3.
[0120] In the modified fibroin according to the present embodiment, a glutamine residue
content rate is preferably 9% or less, more preferably 7% or less, still more preferably
4% or less, and particularly preferably 0%.
[0121] In the present specification, the "glutamine residue content rate" is a value calculated
by the following method.
[0122] In a fibroin (a modified fibroin or a fibroin derived from the natural 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 a case where the total number of glutamine residues included in all REPs
included in a sequence (sequence equivalent to "region A" in Fig. 3) excluding a sequence
from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is denoted by u, and the total number of amino acid residues
in all REPs excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence and further excluding (A)
n motifs is denoted by t, the glutamine residue content rate is calculated as u/t.
For the calculation of the glutamine residue content rate, the "sequence excluding
a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" is used for the same reason described above.
[0123] The domain sequence of the modified fibroin according to the present embodiment may
include an amino acid sequence equivalent to an amino acid sequence in which one or
a plurality of glutamine residues in REP are deleted or substituted with other amino
acid residues, as compared with a fibroin derived from the natural fibroin.
[0124] The "other amino acid residue" may be an amino acid residue other than a glutamine
residue but is preferably an amino acid residue having a higher hydropathy index than
that of a glutamine residue. The hydropathy indices of amino acid residues are as
shown in Table 1.
[0125] As shown in Table 1, amino acid residues having a higher hydropathy index than a
glutamine residue include an amino acid residue selected from 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). Among these, an amino acid residue selected from 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 isoleucine (I), valine (V), leucine
(L), and phenylalanine (F) is still more preferable.
[0126] In the modified fibroin according to the present embodiment, the hydrophobicity of
REP is preferably -0.8 or more, more preferably -0.7 or more, still more preferably
0 or more, even 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,
may be 1.0 or less, and may be 0.7 or less.
[0127] In the present specification, the "hydrophobicity of REP" is a value calculated by
the following method.
[0128] In a fibroin (a modified fibroin or a fibroin derived from the natural 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 a case where the total sum of the hydropathy index of each amino acid residue
included in all REPs included in a sequence (sequence equivalent to "region A" in
Fig. 1) excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence is denoted by v, and the total number of amino acid residues
in all REPs excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence and further excluding (A)
n motifs is denoted by t, the hydrophobicity is calculated as v/t. For the calculation
of the hydrophobicity of REP, the "sequence excluding a sequence from the (A)
n motif located closest to the C-terminal side to the C-terminal of the domain sequence
from the domain sequence" is used for the same reason described above.
[0129] The domain sequence of the modified fibroin according to the present embodiment may
further include an amino acid sequence equivalent to an amino acid sequence in which
one or a plurality of amino acid residues are substituted, deleted, inserted and/or
added, in addition to the modification of the amino acid sequence in which one or
a plurality of glutamine residues in REP are deleted and/or one or a plurality of
glutamine residues in REP are substituted with other amino acid residues, as compared
with a fibroin derived from the natural fibroin.
[0130] The modified fibroin according to the present embodiment can be obtained by, for
example, with respect to a cloned gene sequence of a fibroin derived from the natural
fibroin, deleting one or a plurality of glutamine residues in REP and/or by substituting
one or a plurality of glutamine residues in REP with other amino acid residues. Further,
for example, the modified fibroin may also be obtained by designing an amino acid
sequence equivalent to an amino acid sequence in which with respect to the amino acid
sequence of a fibroin derived from the natural fibroin, one or a plurality of glutamine
residues in REP are deleted and/or one or a plurality of glutamine residues in REP
are substituted with other amino acid residues, and chemically synthesizing a nucleic
acid encoding the designed amino acid sequence.
[0131] A more specific example of the modified fibroin according to the embodiment of the
present invention may be a modified fibroin including (6-i) the amino acid sequence
set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:
31, SEQ ID NO: 38, or SEQ ID NO: 39, or (6-ii) an amino acid sequence having 90% or
more sequence identity with the amino acid sequence set forth in SEQ ID NO: 27, SEQ
ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO:
39.
[0132] The modified fibroin of (6-i) will be described.
[0133] The amino acid sequence (Met-PRT410) set forth in SEQ ID NO: 4 is a modified amino
acid sequence obtained by changing the number of the consecutive alanine residues
in the (A)
n motif to five, or the like, so as to improve productivity, based on the base sequence
and amino acid sequence of Nephila clavipes (GenBank Accession No.: P46804.1, GI:
1174415) which is a fibroin derived from the natural fibroin. However, since Met-PRT410
has no modification of glutamine residue (Q), the glutamine residue content rate thereof
is the same as the glutamine residue content of a fibroin derived from the natural
fibroin.
[0134] The amino acid sequence (M_PRT888) set forth in SEQ ID NO: 27 is obtained by substituting
all QQs in Met-PRT410 (SEQ ID NO: 4) with VLs.
[0135] The amino acid sequence (M_PRT965) set forth in SEQ ID NO: 28 is obtained by substituting
all QQs in Met-PRT410 (SEQ ID NO: 4) with TSs and substituting the remaining Qs with
As.
[0136] The amino acid sequence (M PRT889) set forth in SEQ ID NO: 29 is obtained by substituting
all QQs in Met-PRT410 (SEQ ID NO: 4) with VLs and substituting the remaining Qs with
Is.
[0137] The amino acid sequence (M PRT916) set forth in SEQ ID NO: 30 is obtained by substituting
all QQs in Met-PRT410 (SEQ ID NO: 4) with VIs and substituting the remaining Qs with
Ls.
[0138] The amino acid sequence (M PRT918) set forth in SEQ ID NO: 31 is obtained by substituting
all QQs in Met-PRT410 (SEQ ID NO: 4) with VFs and substituting the remaining Qs with
Is.
[0139] The amino acid sequence (M_PRT525) set forth in SEQ ID NO: 37 is obtained by, with
respect to Met-PRT410 (SEQ ID NO: 4), inserting two alanine residues in a region (A5)
in which alanine residues are consecutive, and by deleting two domain sequences at
the C-terminal side and substituting 13 glutamine residues (Q) with serine residues
(S) or prolines (P) so that the molecular weight thereof is approximately the same
as that of Met-PRT410.
[0140] The amino acid sequence (M PRT699) set forth in SEQ ID NO: 38 is obtained by substituting
all QQs in M_PRT525 (SEQ ID NO: 37) with VLs.
[0141] The amino acid sequence (M PRT698) set forth in SEQ ID NO: 39 is obtained by substituting
all QQs in M_PRT525 (SEQ ID NO: 37) with VLs and substituting the remaining Qs with
Is.
[0142] The glutamine residue content rate of any of the amino acid sequences set forth in
SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 38, and SEQ ID NO: 39 is 9% or less (Table 2).
[Table 2]
Modified fibroin |
Glutamine residue content rate |
GPGXX motif content rate |
Hydrophobicity of REP |
Met-PRT410 (SEQ ID NO: 4) |
17.7% |
27.9% |
-1.52 |
M_PRT888 (SEQ ID NO: 27) |
6.3% |
27.9% |
-0.07 |
M_PRT965 (SEQ ID NO: 28) |
0.0% |
27.9% |
-0.65 |
M_PRT889 (SEQ ID NO: 29) |
0.0% |
27.9% |
0.35 |
M_PRT916 (SEQ ID NO: 30) |
0.0% |
27.9% |
0.47 |
M_PRT918 (SEQ ID NO: 31) |
0.0% |
27.9% |
0.45 |
M_PRT525 (SEQ ID NO: 37) |
13.7% |
26.4% |
-1.24 |
M_PRT699 (SEQ ID NO: 38) |
3.6% |
26.4% |
-0.78 |
M_PRT698 (SEQ ID NO: 39) |
0.0% |
26.4% |
-0.03 |
[0143] The modified fibroin of (6-i) may consist of the amino acid sequence set forth in
SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 38, or SEQ ID NO: 39.
[0144] The modified fibroin of (6-ii) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 38, or SEQ ID NO:
39. The modified fibroin of (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.
[0145] The modified fibroin of (6-ii) preferably has the glutamine residue content rate
of 9% or less. In addition, the modified fibroin of (6-ii) preferably has the GPGXX
motif content rate of 10% or more.
[0146] The above-described modified fibroin may include a tag sequence at either or both
of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect,
and visualize the modified fibroin.
[0147] A more specific example of the modified fibroin including a tag sequence may be a
modified fibroin including (6-iii) the amino acid sequence set forth in SEQ ID NO:
32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or
SEQ ID NO: 41, or (6-iv) an amino acid sequence having 90% or more sequence identity
with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:
34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO: 41.
[0148] The amino acid sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34,
SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, and SEQ ID NO: 41 are respectively amino
acid sequences obtained by adding the amino acid sequence (including a His tag and
a hinge sequence) set forth in SEQ ID NO: 5 to the N-terminal of the amino acid sequences
set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:
31, SEQ ID NO: 38, and SEQ ID NO: 39. Since only the tag sequence is added to the
N-terminal, the glutamine residue content rate are not changed, and any of the amino
acid sequences set forth in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, SEQ ID NO: 36, SEQ ID NO: 40, and SEQ ID NO: 41 has the glutamine residue content
rate of 9% or less (Table 3).
[Table 3]
Modified fibroin |
Glutamine residue content rate |
GPGXX motif content rate |
Hydrophobicity of REP |
PRT888 (SEQ ID NO: 32) |
6.3% |
27.9% |
-0.07 |
PRT965 (SEQ ID NO: 33) |
0.0% |
27.9% |
-0.65 |
PRT889 (SEQ ID NO: 34) |
0.0% |
27.9% |
0.35 |
PRT916 (SEQ ID NO: 35) |
0.0% |
27.9% |
0.47 |
PRT918 (SEQ ID NO: 36) |
0.0% |
27.9% |
0.45 |
PRT699 (SEQ ID NO: 40) |
3.6% |
26.4% |
-0.78 |
PRT698 (SEQ ID NO: 41) |
0.0% |
26.4% |
-0.03 |
[0149] The modified fibroin of (6-iii) may consist of the amino acid sequence set forth
in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ
ID NO: 40, or SEQ ID NO: 41.
[0150] The modified fibroin of (6-iv) includes an amino acid sequence having 90% or more
sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID
NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ ID NO:
41. The modified fibroin of (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.
[0151] The modified fibroin of (6-iv) preferably has the glutamine residue content rate
of 9% or less. In addition, the modified fibroin of (6-iv) preferably has the GPGXX
motif content rate of 10% or more.
[0152] The above-mentioned modified fibroin may include a secretory signal for releasing
the protein produced in the recombinant protein production system to the outside of
a host. The sequence of the secretory signal can be appropriately set depending on
the type of the host.
(Manufacturing method for modified fibroin)
[0153] The modified fibroin (hereinafter, also simply referred to as "protein") according
to the present embodiment can be produced, for example, by expressing a nucleic acid
in a host transformed with an expression vector having the nucleic acid sequence encoding
the modified fibroin and one or a plurality of regulatory sequences operably linked
to the nucleic acid sequence.
[0154] The producing method for a nucleic acid encoding a modified fibroin is not particularly
limited. For example, the nucleic acid is produced by cloning a gene encoding the
natural fibroin by amplification with polymerase chain reaction (PCR) or the like
and modifying the gene by a genetic engineering method, by chemically synthesizing
the nucleic acid. The method for chemically synthesizing a nucleic acid is not particularly
limited, and for example, the gene can be chemically synthesized by a method in which
oligonucleotides are automatically synthesized by AKTA oligopilot plus 10/100 (GE
Healthcare Japan Corporation) or the like and are linked by PCR or the like, based
on the amino acid sequence information of the protein obtained from the NCBI web database
or the like. In this case, in order to facilitate purification and/or confirmation
of the protein, a nucleic acid may be synthesized such that a protein having an amino
acid sequence obtained by adding an amino acid sequence consisting of a start codon
and a His10 tag to the N-terminal of the above amino acid sequence is encoded.
[0155] The regulatory sequence is a sequence (for example, a promoter, an enhancer, a ribosome
binding sequence, or a transcription termination sequence) that controls the expression
of a modified fibroin in a host, and can be appropriately selected depending on the
type of the host. As a promoter, an inducible promoter that functions in a host cell
and is capable of inducing the expression of a modified fibroin may be used. An inducible
promoter is a promoter that can control transcription by the presence of an inducer
(an expression inducer), the absence of a repressor molecule, or physical factors
such as an increase or decrease in temperature, osmotic pressure, or pH value.
[0156] The type of the expression vector such as a plasmid vector, a viral vector, a cosmid
vector, a fosmid vector, or an artificial chromosome vector can be appropriately selected
depending on the type of the host. As the expression vector, an expression vector
that can autonomously replicate in a host cell or can be incorporated into a chromosome
of a host and which contains a promoter at a position capable of transcribing the
nucleic acid that encodes a protein is suitably used.
[0157] Both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal
cells, and plant cells can be suitably used as a host.
[0158] Preferred examples of the prokaryotic host cells include bacteria belonging to the
genus Escherichia, the genus Brevibacillus, the genus Serratia, the genus Bacillus,
the genus Microbacterium, the genus Brevibacterium, the genus Corynebacterium, and
the genus Pseudomonas. Examples of microorganisms belonging to the genus Escherichia
include Escherichia coli. Examples of the microorganisms belonging to the genus Brevibacillus
include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia
include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus
include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium
include Microbacterium ammoniaphilum. Examples of microorganisms belonging to the
genus Brevibacterium 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.
[0159] In a case where a prokaryote is used as a host, examples of a vector into which a
nucleic acid encoding a protein is introduced include pBTrp2 (manufactured by Boehringer
Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b,
pCold, pUB110, and pNCO2 (Japanese Unexamined Patent Publication No.
2002-238569).
[0160] Examples of eukaryotic hosts include yeast and filamentous fungi (mold and the like).
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.
[0161] In a case where a eukaryote is used as a host, examples of the vector into which
a nucleic acid encoding a modified fibroin is introduced include YEp13 (ATCC37115)
and YEp24 (ATCC37051). As a method for introducing an expression vector into the above
host cell, any method can be used as long as the method introduces DNA into the host
cell. Examples thereof include a method using calcium ions [
Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate
method, and competent method.
[0162] As for the method for expressing a nucleic acid using a host transformed with an
expression vector, secretory production, fusion protein expression, or the like, in
addition to the direct expression, can be carried out according to the method described
in
Molecular Cloning, 2nd edition.
[0163] The modified fibroin can be produced, for example, by culturing a host transformed
with the expression vector in a culture medium, producing and accumulating the protein
in the culture medium, and then collecting the modified fibroin from the culture medium.
The method for culturing a host in a culture medium can be carried out according to
a method commonly used for culturing a host.
[0164] In the case where the host is a prokaryote such as Escherichia coli or a eukaryote
such as yeast, any of a natural medium and a synthetic medium may be used as a culture
medium of the host as long as the medium contains a carbon source, a nitrogen source,
inorganic salts and the like which can be utilized by the host and the medium can
be used for efficiently culturing the host.
[0165] As the carbon source, any carbon source that can be utilized by the transformed microorganism
may be used. Examples of the carbon source that can be utilized include carbohydrates
such as glucose, fructose, sucrose, and molasses, starch and starch hydrolyzates containing
them, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol
and propanol. Examples of the nitrogen source that can be utilized include ammonium
salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate,
ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone,
meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean cake and
soybean cake hydrolyzate, and various fermented microbial cells and digested products
thereof. Examples of the inorganic salt that can be utilized include potassium dihydrogen
phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride,
ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
[0166] Culture of a prokaryote such as Escherichia coli or a eukaryote such as yeast can
be carried out under aerobic conditions such as shaking culture or deep aeration stirring
culture. The culture temperature is, for example, 15°C to 40°C. The culture time is
usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium
during the culture at 3.0 to 9.0. The pH of the culture medium can be adjusted using
an inorganic acid, an organic acid, an alkali solution, urea, calcium carbonate, ammonia,
or the like.
[0167] In addition, antibiotics such as ampicillin and tetracycline may be added to the
culture medium as necessary during the culture. In a case of culturing a microorganism
transformed with an expression vector using an inducible promoter as a promoter, an
inducer may be added to the medium as necessary. For example, in a case of culturing
a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside
or the like is used, and in a case of culturing a microorganism transformed with an
expression vector using a trp promoter, indole acrylic acid or the like may be added
to the medium.
[0168] The expressed protein can be isolated and purified by a commonly used method. For
example, in a case where the protein is expressed in a dissolved state in cells, the
host cells are recovered by centrifugation after the completion of the culture, suspended
in an aqueous buffer solution, and then disrupted using an ultrasonicator, a French
press, a Manton-Gaulin homogenizer, a Dyno-Mill, or the like to obtain a cell-free
extract. From the supernatant obtained by centrifuging the cell-free extract, a purified
preparation can be obtained by a method commonly used for protein isolation and purification,
that is, 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
or DIAION HPA-75 (manufactured by Mitsubishi Kasei Kogyo Kabushiki Kaisha), an cation
exchange chromatography method using a resin such as S-Sepharose FF (manufactgured
by Pharmacia Corporation), a hydrophobic chromatography method using a resin such
as butyl sepharose or phenyl sepharose, a gel filtration method using a molecular
sieve, an affinity chromatography method, a chromatofocusing method, or an electrophoresis
method such as isoelectric focusing or the like, using the above methods singly or
in combination thereof.
[0169] In addition, in a case where the protein is expressed to form an insoluble body in
the cell, similarly, the host cells are recovered, disrupted and centrifuged to recover
the insoluble body of the protein as a precipitated fraction. The recovered insoluble
body of the protein can be solubilized with a protein denaturing agent. After this
operation, a purified preparation of the protein can be obtained by the same isolation
and purification method as described above. In a case where the protein is secreted
extracellularly, the protein can be recovered from the culture supernatant. That is,
a culture supernatant is obtained by treating the culture by a technique such as centrifugation,
and a purified preparation can be obtained from the culture supernatant by using the
same isolation and purification method as described above.
(Manufacturing method for artificial fibroin filament)
[0170] An artificial fibroin filament (hereinafter, sometimes referred to as "artificial
protein filament" or simply "protein fiber") may include other protein or a contaminant
as long as it includes the modified fibroin as the main component. The artificial
fibroin filament is preferably an artificial spider silk fibroin filament. The artificial
fibroin filament can be manufactured by a known spinning method. That is, for example,
in a case of manufacturing a protein filament including a modified fibroin as the
main component, first, the modified fibroin produced according to the above-mentioned
method is added to a solvent such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide
(DMF), formic acid, or hexafluoroisopropanol (HFIP), as necessary, together with an
inorganic salt as a dissolution accelerator and dissolved to prepare a doping liquid.
Then, using the doping liquid, spinning can be performed by a known spinning method
such as wet-type spinning, dry-type spinning, dry-wet-type spinning, or melt spinning
to obtain the protein filament. A preferred spinning method is wet-type spinning and
dry-wet-type spinning.
[0171] Fig. 3 is an explanatory view schematically illustrating one example of a spinning
device for manufacturing a protein filament. A spinning device 10 shown in Fig. 3
is an example of a spinning device for dry-wet-type spinning and includes an extrusion
device 1, an undrawn yarn manufacturing device 2, a wet heat drawing device 3, and
a drying device 4.
[0172] A spinning method using the spinning device 10 will be described. First, a doping
liquid 6 stored in a storage tank 7 is pushed out from a spinneret 9 by a gear pump
8. In the laboratory scale, the doping liquid may be filled in a cylinder and extruded
from a nozzle using a syringe pump. Next, the extruded doping liquid 6 is supplied
into a coagulation liquid 11 in a coagulation liquid bath 20 via an air gap 19, the
solvent is removed, the modified fibroin is coagulated, and a fibrous coagulate is
formed. Then, the fibrous coagulate is supplied into a warm water 12 in a drawing
bath 21 and is drawn. A drawing ratio is determined according to a speed ratio of
a supply nip roller 13 to a withdrawing nip roller 14. Thereafter, the drawn fibrous
coagulate is supplied to a drying device 4 and dried in a yarn path 22, and the protein
filament 36 is obtained as a wound yarn body 5. Reference signs 18a to 18g indicate
yarn guides.
[0173] The coagulation liquid 11 may be any solvent that can be desolvated, and examples
thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol,
and 2-propanol, and acetone. The coagulation liquid 11 may appropriately contain water.
The temperature of the coagulation liquid 11 is preferably 0°C to 30°C. In a case
where a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the
spinneret 9, the extrusion speed is preferably 0.2 to 6.0 mL/hour per hole and more
preferably 1.4 to 4.0 mL/hour. The distance that the coagulated modified fibroin passes
through the coagulation liquid 11 (substantially, the distance from the yarn guide
18a to the yarn guide 18b) may be a length that allows efficient desolvation, for
example, 200 to 500 mm. The withdrawing speed of the undrawn yarn may be, for example,
1 to 20 m/min and preferably 1 to 3 m/min. The residence time in the coagulation liquid
11 may be, for example, 0.01 to 3 minutes and preferably 0.05 to 0.15 minutes. In
addition, drawing (pre-drawing) may be performed in the coagulation liquid 11. The
coagulation liquid bath 20 may be provided in multiple stages, and the drawing may
be performed in each stage or in a specific stage as necessary.
[0174] As the drawing performed in a case of obtaining the protein filament, for example,
a pre-drawing performed in the coagulation liquid bath 20 and a wet heat drawing performed
in the drawing bath 21 are employed, and a dry heat drawing is also employed.
[0175] The wet heat drawing can be performed in warm water, in a solution obtained by adding
an organic solvent or the like to warm water, or in heated steam. The temperature
may be, for example, 50°C to 90°C and preferably 75°C to 85°C. In the wet heat drawing,
the undrawn yarn (or pre-drawn yarn) can be drawn, for example, by 1 to 10 times and
preferably by 2 to 8 times.
[0176] The dry heat drawing can be performed using an electric tubular furnace, a dry heat
plate, or the like. The temperature may be, for example, 140°C to 270°C and preferably
160°C to 230°C. In the dry heat drawing, the undrawn yarn (or pre-drawn yarn) can
be drawn, for example, by 0.5 to 8 times and preferably by 1 to 4 times.
[0177] The wet heat drawing and the dry heat drawing may be performed independently or in
combination or may be performed in multiple stages. That is, the wet heat drawing
and the dry heat drawing can be performed in a suitable combination, for example,
in a manner in which a first stage drawing is performed by wet heat drawing and a
second stage drawing is performed by dry heat drawing or in a manner in which the
first stage drawing is performed by wet heat drawing, the second stage drawing is
performed by wet heat drawing, and a third stage drawing is performed by dry heat
drawing.
[0178] The lower limit value of the final drawing ratio with respect to the undrawn yarn
(or pre-drawn yarn) is preferably any of 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, 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.
[0179] The length of the protein filament obtained by the above method can be suitably adjusted
depending on the spinning conditions and is preferably more than 1,500 m, and may
be 10,000 m or more, 15,000 m or more, or 20,000 m or more.
[0180] The artificial fibroin filament preferably has a shrinkage rate (shrinkage rate after
drying) of more than 7% and may have a shrinkage rate of 10% or more, 15% or more,
25% or more, 32% or more, 40% or more, 48% or more, 56% or more, 64% or more, or 72%
or more, when the artificial fibroin filament has been brought into contact with an
aqueous medium to be described later (for example, water having a temperature lower
than the boiling point of the water) and then dried. The shrinkage rate after drying
is usually 80% or less. Here, the shrinkage rate after drying of the fibroin filament
is defined by the following expression.
[0181] In addition, the artificial fibroin filament may have a shrinkage rate (shrinkage
rate when wetted) of, for example, 2% or more and may have a shrinkage rate of 2.5%
or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more,
or 6% or more, when the artificial fibroin filament has been brought into a wet state
by bringing the artificial fibroin filament into contact with an aqueous medium to
be described later (for example, water having a temperature lower than the boiling
point of the water). The upper limit of the shrinkage rate when wetted is not particularly
limited but is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7%
or less, 6% or less, 5% or less, 4% or less, or 3% or less. Here, the shrinkage rate
when wetted can be calculated by the following expression.
<Process b>
[0182] The process b (cutting process) is a process of cutting the artificial fibroin filament
to obtain an artificial fibroin staple. Here, in a case where the process b is performed
before process c, the artificial fibroin filament to be cut is an artificial fibroin
filament before being crimped, and in a case where the process b is performed after
the process c, the artificial fibroin filament to be cut is an artificial fibroin
filament after being crimped.
[0183] The cutting can be performed using any device capable of cutting a protein fiber.
An example of such a cutting device includes a tabletop-type fiber cutting machine
(s/NO. IT-160201-NP-300).
[0184] The length of the staple is not particularly limited, but is preferably 20 mm or
more, and may be 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.
<Process c>
[0185] The process c (crimping process) is a process of bringing the artificial fibroin
filament or the artificial fibroin staple into contact with an aqueous medium and
then crimping the artificial fibroin filament or the artificial fibroin staple (hereinafter
sometimes referred to as "water crimping"). Here, in a case where the process c is
performed before the process b, the artificial fibroin filament is crimped by being
brought into contact with water to be curled, and in a case where the process c is
performed after the process b, the artificial fibroin staple obtained by cutting the
artificial fibroin filament is crimped by being brought into contact with water to
be curled.
[0186] By being brought into contact with an aqueous medium, the artificial fibroin filament
or the artificial fibroin staple can be crimped without depending on the external
force. The aqueous medium is a medium of a liquid or gas (steam) containing water
(including steam). The aqueous medium may be water or a mixed liquid of water and
a hydrophilic solvent. Further, as the hydrophilic solvent, for example, a volatile
solvent such as ethanol and methanol or a vapor thereof can be used. The aqueous medium
may be a mixed liquid of water and a volatile solvent such as ethanol or methanol
and is preferably water or a mixed liquid of water and ethanol. By using an aqueous
medium containing a volatile solvent or a vapor thereof, it is possible to improve
the drying speed after the water crimping, and further there is a possibility of imparting
a soft texture to the crimped staple finally obtained. The ratio of water to the volatile
solvent or the vapor thereof is not particularly limited, and, for example, water:volatile
solvent or vapor thereof may be 10:90 to 90:10 by mass ratio. The proportion of water
is preferably 30% by mass or more and may be 40% by mass or 50% by mass or more. In
a case where the aqueous medium is a liquid, it is preferable to disperse an oil agent
in the aqueous medium. In this case, the water crimping and the oil agent adhering
can be performed at the same time. As the oil agent, any oil agent can be used as
long it is a known oil agent used for general purposes including process passability
and function impartability, such as an antistatic property, a friction reduction property,
a flexibility imparting property, and a water repellency imparting property. The amount
of the oil agent is not particularly limited and may be, for example, 1% to 10% by
mass or 2% to 5% by mass with respect to the total amount of the oil agent and the
aqueous medium.
[0187] The aqueous medium is preferably a liquid or gas containing water and having a temperature
of 10°C 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,
more preferably 100 to 120°C. In a case where the steam of the aqueous medium is 230°C
or less, the heat denaturation of the protein filament can be prevented. In a case
where 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 from the viewpoint of efficiently
imparting crimpness, and is preferably 60°C or lower from the viewpoint of highly
maintaining fiber strength of the protein filament.
[0188] The time of bringing into contact with the aqueous medium is not particularly limited,
but may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and preferably
10 minutes or shorter from the viewpoint of productivity. In the case of steam, it
is considered that a high shrinkage rate can be obtained in a short time in comparison
with a liquid. The contact with the aqueous medium may be carried out under normal
pressure or under reduced pressure (for example, vacuum).
[0189] As a method for bringing into contact with the aqueous medium, a method of immersing
an artificial fibroin filament or artificial fibroin staple in the aqueous medium,
a method of spraying the steam of the aqueous medium to an artificial fibroin filament
or artificial fibroin staple, and a method of exposing an artificial fibroin filament
or an artificial fibroin staple to the environment filled with the steam of the aqueous
medium. In a case where the aqueous medium is steam, bringing an artificial fibroin
filament or an artificial fibroin staple into contact with the aqueous medium can
be performed using a general steam setting device. Specific examples of the steam
setting device include a device of product name: FMSA type steam setter (manufactured
by Fukushin Kyougyo Co., Ltd.) and a device of a product name: EPS-400 (manufactured
by Tsujii Dyeing Machine Manufacturing Co., Ltd.). The specific example of the method
for crimping an artificial fibroin filament or an artificial fibroin staple by using
the steam of the aqueous medium includes a method of accommodating an artificial fibroin
filament or an artificial fibroin staple in a predetermined accommodating chamber
and bringing the artificial fibroin filament or the artificial fibroin staple into
contact with steam, while introducing the steam of the aqueous medium into the accommodating
chamber and adjusting the temperature inside the accommodating chamber to the above-described
predetermined temperature (for example, 100°C to 230°C).
[0190] The crimping process of an artificial fibroin filament or an artificial fibroin staple
by contact with the aqueous medium is preferably performed in a state in which no
tensile force is applied to the artificial fibroin filament or the artificial fibroin
staple (not tensioned in the fiber axis direction) or in a state in which a predetermined
amount of tensile force is applied (tensioned by a predetermined amount in the fiber
axis direction). At this time, the degree of crimpness can be controlled by adjusting
the tensile force applied to the artificial fibroin filament or the artificial fibroin
staple. Examples of the adjusting method for the tensile force applied to the artificial
fibroin filament or the artificial fibroin staple include, a method in which the artificial
fibroin filaments or the artificial fibroin staples are, for example, hung by weighting
units having various weights, and the loads applied to the filaments and staples are
adjusted, a method in which both ends of the filament or staple are fixed in a state
in which the filament or the staple are slackened, and the amount of slackness are
changed variously, and a method in which the filament is wound around a wound body
such as a paper tube or a bobbin so that the winding force is suitably changed (tightening
force on the paper tube or a bobbin).
[0191] Further, the artificial fibroin filament or the artificial fibroin staple may be
dried after being brought into contact with the aqueous medium (for example, water
having a temperature lower than the boiling point of the water). The drying method
is not particularly limited, and the drying may be natural drying, hot air drying,
or hot roller drying. The drying temperature is not particularly limited and may be,
for example, 20°C to 150°C, preferably 40°C to 120°C, and more preferably 60°C to
100°C.
[0192] The artificial fibroin filament or the artificial fibroin staple after crimping and
then drying preferably has a shrinkage rate (shrinkage rate after drying) of more
than 7% and may have a shrinkage rate of 10% or more, 15% or more, 25% or more, 32%
or more, 40% or more, 48% or more, 56% or more, 64% or more, or 72% or more. The shrinkage
rate after drying is usually 80% or less. Here, the shrinkage rate after drying can
be calculated by the following expression.
[0193] In addition, the artificial fibroin filament or the artificial fibroin staple may
have a shrinkage rate (shrinkage rate when wetted) of, for example, 2% or more and
may have a shrinkage rate of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5%
or more, 5% or more, 5.5% or more, or 6% or more, when the artificial fibroin filament
has been brought into a wet state by bringing the artificial fibroin filament into
contact with an aqueous medium (or example, water having a temperature lower than
the boiling point of the water). The upper limit of the shrinkage rate when wetted
is not particularly limited but is 80% or less, 60% or less, 40% or less, 20% or less,
10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less. Here,
the shrinkage rate when wetted can be calculated by the following expression.
[0194] The process b and the process c may be performed in a batch system or a continuous
system. In the case of a batch system, for example, the artificial fibroin staple
obtained by cutting the artificial fibroin filament is put into a container containing
an aqueous medium at an appropriate temperature, and after being brought into contact
therewith for a certain time, and then taken out and dried. In the case of a continuous
system, for example, while sending out the filament from a bobbin around which the
artificial fibroin filament wound, after being immersed in an aqueous medium at an
appropriate temperature, the filament is dried by blowing hot air or sending the filament
out on a hot roller, and then cut continuously.
Use applications for protein crimped staple
[0195] The obtained protein crimped staple is a single fiber having a soft feel and can
be used for manufacturing a composite material such as spun yarn, nonwoven fabric,
and a composite.
[0196] A predetermined amount of the artificial fibroin fiber can be shrunk when first brought
into contact with an aqueous medium after spinning. Since the protein crimped staple
obtained by the manufacturing method of the present invention has already been brought
into contact with moisture (aqueous medium), it is possible to suppress the dimensional
change (shrinkage) of the staple due to moisture absorption during storage after manufacturing
the staple or during the manufacturing process for a product using the staple.
Examples
[0197] Hereinafter, the present invention will be described more specifically based on Examples.
However, the present invention is not limited to the following Examples.
<Manufacturing example of artificial spider silk protein (artificial spider silk fibroin)
filament>
(1) Preparation of plasmid expressing strain
[0198] Based on the base sequence and the amino acid sequence of a fibroin (GenBank Accession
No.: P46804.1, GI: 1174415) derived from Nephila clavipes, a modified fibroin having
the amino acid sequence set forth in SEQ ID NO: 13 (hereinafter, also referred to
as "PRT799") was designed. The amino acid sequence set forth in SEQ ID NO: 13 has
an amino acid sequence obtained by substituting, inserting, and deleting an amino
acid residue for the purpose of improving productivity with respect to the amino acid
sequence of the fibroin derived from Nephila clavipes, and furthermore, the amino
acid sequence set forth in SEQ ID NO: 5 (tag sequence and hinge sequence) is added
to the N-terminal of the sequence.
[0199] Next, a nucleic acid encoding PRT799 was synthesized. In the nucleic acid, an Ndel
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). Thereafter, the nucleic
acid was enzymatically cleaved by treatment with NdeI and EcoRI, and then recombinated
into a protein expression vector pET-22b (+) to obtain an expression vector.
(2) Expression of protein
[0200] Escherichia coli BLR (DE3) was transformed with a pET22b(+) expression vector including
the nucleic acid encoding a protein having the amino acid sequence set forth in SEQ
ID NO: 13. The transformed Escherichia coli was cultured in 2 mL of an LB medium containing
ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture
medium (Table 4) containing ampicillin so that the OD600 was 0.005. While maintaining
the temperature of the culture solution at 30°C, flask culturing was carried out (for
about 15 hours) until the OD600 reached 5, thereby obtaining a seed culture solution.
[Table 4]
Seed culture medium |
Reagent |
Concentration (g/L) |
Glucose |
5.0 |
KH2PO4 |
4.0 |
K2HPO4 |
9.3 |
Yeast Extract |
6.0 |
Ampicillin |
0.1 |
[0201] The seed culture solution was added to a jar fermenter containing 500 mL of a production
medium (Table 5) so that the OD600 was 0.05. The culture was carried out while keeping
the culture solution temperature at 37°C and controlling the pH constant at 6.9. Further,
the concentration of dissolved oxygen in the culture solution was maintained at 20%
of the dissolved oxygen saturation concentration.
[Table 5]
Production medium |
Reagent |
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 |
GD-113 (anti-foaming agent) |
0.1(mL/L) |
[0202] Immediately after glucose in the production medium was completely consumed, a feed
solution (455 g/1L of glucose and 120 g/1L of Yeast Extract) was added at a rate of
1 mL/min. The culture was carried out while keeping the culture solution temperature
at 37°C and controlling the pH constant at 6.9. Further, the concentration of dissolved
oxygen in the culture solution was maintained at 20% of the dissolved oxygen saturation
concentration, and the culture was carried out for 20 hours. Thereafter, 1 M isopropyl-β-thiogalactopyranoside
(IPTG) was added to the culture solution to a final concentration of 1 mM to induce
the expression of the modified fibroin. 20 hours after the addition of IPTG, the culture
solution was centrifuged to recover the bacterial cell pellet. SDS-PAGE was carried
out using bacterial cell pellets prepared from the culture solution before the addition
of IPTG and after the addition of IPTG, and the expression of the target modified
fibroin was checked by the IPTG addition-dependent appearance of a band equivalent
to the size of the target modified fibroin.
(3) Purification of protein
[0203] The bacterial cell pellet recovered 2 hours after the addition of IPTG was washed
with a 20 mM Tris-HCl buffer solution (pH 7.4). The bacterial cell pellet after washing
was suspended in 20 mM Tris-HCl buffer solution (pH 7.4) containing about 1 mM PMSF,
and the cell suspension was disrupted with a high-pressure homogenizer (manufactured
by GEA Niro Soavi SpA). The disrupted cells were centrifuged to obtain a precipitate.
The obtained precipitate was washed with a 20 mM Tris-HCl buffer solution (pH 7.4)
until the obtained precipitate became highly pure. The precipitate after washing was
suspended in 8 M guanidine buffer solution (8 M guanidine hydrochloride, 10 mM sodium
dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so that the concentration
of the suspension was 100 mg/mL, and dissolved by stirring with a stirrer at 60°C
for 30 minutes. After dissolution, dialysis was carried out in water using a dialysis
tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white protein
aggregate obtained after dialysis was recovered by centrifugation, the water content
was removed with a lyophilizer, and the lyophilized powder was recovered to obtain
the modified spider silk fibroin "PRT799".
(4) Manufacturing of protein filament
[0204] The above-described modified fibroin (PRT799) was added to DMSO such that the concentration
was 24% by mass, and then LiCl was added thereto as a dissolution accelerator such
that the concentration was 4.0% by mass. Then, the modified fibroin was dissolved
for 3 hours using a shaker to obtain a DMSO solution of the modified fibroin. Dust
and bubbles in the obtained DMSO solution were removed to obtain a doping liquid.
The solution viscosity of the doping liquid was 5,000 cP (centipoise) at 90°C.
[0205] Known dry-wet-type spinning was performed using the doping liquid obtained as described
above and the spinning device 10 shown in Fig. 4, and the artificial spider silk fibroin
fiber was wound around a bobbin. Here, the dry-wet-type spinning was performed under
the following conditions.
Temperature of coagulation liquid (methanol): 5°C to 10°C
Drawing ratio: 4.52 times
Drying temperature: 80°C
<Example 1>
[0206] An artificial spider silk protein staple was prepared by bundling a plurality of
artificial spider silk filaments had been obtained in the manufacturing example of
an artificial spider silk protein and had been wound around a bobbin, and by cutting
the filament into a length of 40 mm by a table-top type fiber cutting machine. The
prepared artificial spider silk protein staple was crimped by being immersed in water
at 40°C for 1 minute to be curled and then dried at 40°C for 18 hours to obtain a
crimped staple. The obtained crimped staple is shown in Fig. 5. The shrinkage rate
of the artificial spider silk protein staple when immersed in water was 50%.
<Example 2>
[0207] An artificial spider silk protein staple was prepared by bundling a plurality of
artificial spider silk filaments had been obtained in the manufacturing example of
an artificial spider silk protein and had been wound around a bobbin, and by cutting
the filament into a length of 40 mm by a table-top type fiber cutting machine. A commercially
available antistatic oil agent was dispersed in water to a concentration of 1% by
weight (concentration in the oil agent dispersion liquid) to obtain an oil agent dispersion
liquid. The prepared artificial spider silk protein staple was crimped by being immersed
in an oil agent dispersion liquid at 20°C for 1 minute to be curled and then dried
at 40°C for 18 hours to obtain a crimped staple. The obtained crimped staple is shown
in Fig. 6. The shrinkage rate of the artificial spider silk protein staple when immersed
in the oil agent dispersion liquid was 50%.
<Comparative Example>
[0208] An artificial spider silk protein filament was simply cut to obtain an uncrimped
staple. A photograph of the obtained uncrimped staple is shown in Fig. 7.
<Example 3>
[0209] An artificial spider silk protein staple was prepared by bundling a plurality of
artificial spider silk filaments had been obtained in the manufacturing example of
an artificial spider silk protein and had been wound around a bobbin, and by cutting
the filament into a length of 40 mm by a table-top type fiber cutting machine. The
prepared artificial spider silk protein staple was crimped by being immersed in a
liquid mixture of water and methanol (50% by mass of a methanol concentration) at
20°C for 1 minute to be curled and then dried at 40°C for 18 hours to obtain a crimped
staple.
[0210] The touch feeling of each of the crimped staple of Example 1 obtained by immersing
in 100% water and the crimped staple of Example 3 obtained by immersing in the liquid
mixture of water and methanol was confirmed by a sensitivity response test. It has
been found that the crimped staple of Example 3 obtained by immersing in the liquid
mixture of water and methanol has a softer feel.
Reference Signs List