title: Secretory production of recombinant human chymase as an active form in Pichia pastoris.
abstract: 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.
authors.affiliation: Drug Discovery Laboratories, Yoshitomi Pharmaceutical Industries Ltd, 2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan. nakakubo@yoshitomi.co.jp
authors.forename: H
authors.initials: H
authors.lastname: Nakakubo
authors.forename: H
authors.initials: H
authors.lastname: Fukuyama
authors.forename: M
authors.initials: M
authors.lastname: Nakajima
authors.forename: T
authors.initials: T
authors.lastname: Imada
authors.forename: S
authors.initials: S
authors.lastname: Uno
authors.forename: N
authors.initials: N
authors.lastname: Shiota
authors.forename: S
authors.initials: S
authors.lastname: Takai
authors.forename: M
authors.initials: M
authors.lastname: Miyazaki
authors.forename: N
authors.initials: N
authors.lastname: Nakamura
mesh_terms: D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D053818:Chymases; D003470:Culture Media; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000804:Angiotensin II; D000803:Angiotensin I; D012697:Serine Endopeptidases; D053818:Chymases