Expression of Recombinant Human Mast Cell Chymase With Asn-Linked Glycans in Glycoengineered Pichia Pastoris
Document Type
Article
Publication Date
1-1-2014
Description
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 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 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.
Citation Information
Smith, Eliot T.; Perry, Evan T.; Sears, Megan B.; and Johnson, David A.. 2014. Expression of Recombinant Human Mast Cell Chymase With Asn-Linked Glycans in Glycoengineered Pichia Pastoris. Protein Expression and Purification. Vol.102 69-75. https://doi.org/10.1016/j.pep.2014.08.005 PMID: 25131858 ISSN: 1046-5928