ArticleProtein expression and purification2014
Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris.
Article in Protein expression and purification, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
5 citing papers in PubMed, 18 citations in OpenAlex.
- The Impact of Methanol Concentration on Recombinant Protein Glycosylation in Pichia pastoris SuperMan5.Microbial biotechnology · 2025Article
- High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.Protein expression and purification · 2023Article
- Influence of carbon source on cell size and production of anti LDL (-) single-chain variable fragment by a recombinant Pichia pastoris strain.Molecular biology reports · 2019Article
- Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison.Microbial cell factories · 2018Article
- Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules.Human vaccines & immunotherapeutics · 2017Review
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
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.
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