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Protein Expr Purif. 2014 Oct;102:69-75. doi: 10.1016/j.pep.2014.08.005. Epub 2014 Aug 12.

Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris.

Author information

1
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Box 70582, Johnson City, TN 37614-1708, USA.
2
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Box 70582, Johnson City, TN 37614-1708, USA. Electronic address: davidj@etsu.edu.

Abstract

Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man)5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5mg 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 KM, whereas heparin effects on kcat 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.

KEYWORDS:

Chymase; Enzyme kinetics; Fermentation; Glycosylation; Pichia pastoris; Serine protease

PMID:
25131858
PMCID:
PMC4165714
DOI:
10.1016/j.pep.2014.08.005
[Indexed for MEDLINE]
Free PMC Article

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