title: Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris.
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.
authors.affiliation: Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Box 70582, Johnson City, TN 37614-1708, USA.
authors.forename: Eliot T
authors.initials: ET
authors.lastname: Smith
authors.affiliation: Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Box 70582, Johnson City, TN 37614-1708, USA.
authors.forename: Evan T
authors.initials: ET
authors.lastname: Perry
authors.affiliation: Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Box 70582, Johnson City, TN 37614-1708, USA.
authors.forename: Megan B
authors.initials: MB
authors.lastname: Sears
authors.affiliation: 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.
authors.forename: David A
authors.initials: DA
authors.lastname: Johnson
mesh_terms: D060846:Cell Engineering; D053818:Chymases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D006031:Glycosylation; D006801:Humans; D008407:Mast Cells / Q000201:enzymology*; D010843:Pichia / Q000235:genetics* / Q000502:physiology; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D053818:Chymases
keywords: Chymase; Enzyme kinetics; Fermentation; Glycosylation; Pichia pastoris; Serine protease