期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 102, 期 -, 页码 69-75出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2014.08.005
关键词
Chymase; Serine protease; Glycosylation; Fermentation; Pichia pastoris; Enzyme kinetics
类别
资金
- ETSU Honors College
- ETSU Office of Research and Sponsored Programs
- NHLB Grant [R15HL091770]
Recombinant human mast cell chymase (rhChyrnase) was expressed in secreted form as an active enzyme in the SuperMan(5) strain of GlycoSwitch (R) Pichia pastoris, which is engineered to produce proteins with (Man)(5)(GlcNAc)(2) Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5 mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDS-PAGE as a single band of 30 kDa and treatment with peptide N-glycosidase F decreased this to 25 kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced K-M, whereas heparin effects on k(cat) were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns. (C) 2014 Elsevier Inc. All rights reserved.
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