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

We succeeded in expressing in a Pichia pastoris (P. pastoris) host a cDNA e ncoding a mature human chymase (h-chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh-chymase) was purif ied from the culture medium via a single one-step heparin-agarose column ch romatography 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 we ight 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 of rh-chymase against Suc-AAPF-p NA was approximately 5.1 mM, which is close to that of purified h-chymase. Copyright (C) 2000 John Wiley & Sons, Ltd.