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Secretory production of recombinant human chymase as an active form in Pichia pastoris
Author(s) -
Nakakubo Hiroshi,
Fukuyama Hajime,
Nakajima Masahide,
Imada Teruaki,
Uno Shusei,
Shiota Naotaka,
Takai Shinji,
Miyazaki Mizuo,
Nakamura Norifumi
Publication year - 2000
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/1097-0061(20000315)16:4<315::aid-yea527>3.0.co;2-4
Subject(s) - pichia pastoris , chymase , biology , recombinant dna , microbiology and biotechnology , biochemistry , polyacrylamide gel electrophoresis , gel electrophoresis , complementary dna , affinity chromatography , agarose , enzyme , gene
We succeeded in expressing in a Pichia pastoris ( P. pastoris ) host a cDNA encoding a mature human chymase (h‐chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh‐chymase) was purified from the culture medium via a single one‐step heparin–agarose column chromatography tracing, using succinyl‐Ala‐Ala‐Pro‐Phe‐para‐nitroanilide (Suc‐AAPF‐pNA) hydrolysing activity. On SDS–polyacrylamide gel electrophoresis (SDS–PAGE), the rh‐chymase showed a diffused protein band with molecular weight of 32–37 kDa. After deglycosylation, however, rh‐chymase changed to a sharp protein band with molecular weight 28 kDa, which is equal in size to deglycosylated h‐chymase. The rh‐chymase had an activity to convert one of the natural substrates, angiotensin I, to angiotensin II. Double reciprocal plot analysis revealed that the K m value ofrh‐chymase against Suc‐AAPF‐pNA was approximately 5.1 m M , which is close to that of purified h‐chymase. Copyright © 2000 John Wiley & Sons, Ltd.