Secretory production of recombinant human chymase as an active form in Pichia pastoris

✍ Scribed by Hiroshi Nakakubo; Hajime Fukuyama; Masahide Nakajima; Teruaki Imada; Shusei Uno; Naotaka Shiota; Shinji Takai; Mizuo Miyazaki; Norifumi Nakamura

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 140 KB
• Volume: 16
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/1097-0061(20000315)16:4<315::aid-yea527>3.0.co;2-4

✦ Synopsis

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 mM, which is close to that of purified h-chymase.