(19)
(11) EP 1 258 527 A8

(12) CORRECTED EUROPEAN PATENT APPLICATION

(48) Corrigendum issued on:
02.04.2003 Bulletin 2003/14

(43) Date of publication:
20.11.2002 Bulletin 2002/47

(21) Application number: 01201616.8

(22) Date of filing: 02.05.2001
(51) International Patent Classification (IPC)7: C12N 9/16, C07K 1/16, C07K 16/40
(84) Designated Contracting States:
AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR
Designated Extension States:
AL LT LV MK RO SI

(71) Applicants:
  • SK Corporation
    Seoul 110-110 (KR)
  • Kim, Dae-Kyong
    Seocho-ku, Seoul 137-069 (KR)

(72) Inventors:
  • Kim, Dae-Kyong
    Seocho-ku, Seoul 137-069 (KR)
  • Jung, Sung-Yun
    Munheung-dong, Buk-ku, Kwangju-si500-110 (KR)
  • Jung, Kwang-Mook
    Bundang-ku, Sungnam-si (KR)

(74) Representative: Long, Giorgio et al
Jacobacci & Partners S.p.A., Via Senato 8
20121 Milano
20121 Milano (IT)

   


(54) Membrane-associated sphingomyelinase derived from bovine brain, isolation method therefor and anti-sphingomyelinase monoclonal antibody


(57) Disclosed is a sphingomyelinase derived from the neuron membrane of the bovine brain, which is 60 kDa in molecular weight and Mg2+-dependent in activity with an optimal pH ranging from 6.0 to 9.0. It is prepared by homogenizing a bovine brain tissue and centrifuging the homogenate to remove cell debris and nuclei; separating cell membranes from the supernatant into a pellet through centrifugation; treating the cell membrane pellet with a buffer containing ammonium sulfate to disassociate the sphingomyelinase from the membrane; subjecting the extract to anion exchange chromatography; sonicating the chromatography fraction in a buffer containing Triton X-100 and centrifuging the sonicated fraction to obtain a supernatant containing the sphingomyelinase; and purifying the sphingomyelinase by subjecting the supernatant to hydrophobic interaction chromatography, anion exchange high-performance liquid chromatography, hydrophobic interaction high performance liquid chromatography, and cation exchange fast protein liquid chromatography, in due order. Also, disclosed is a monoclonal antibody against the sphingomyelinase, produced by Hybridoma SM1A5 (Deposition No. KCLRF-BT-00025).