کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8360440 | 1542339 | 2014 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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 5Â mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDS-PAGE as a single band of 30Â kDa and treatment with peptide N-glycosidase F decreased this to 25Â 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 102, October 2014, Pages 69-75
Journal: Protein Expression and Purification - Volume 102, October 2014, Pages 69-75
نویسندگان
Eliot T. Smith, Evan T. Perry, Megan B. Sears, David A. Johnson,