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High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein

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Indexed by:期刊论文

Date of Publication:2013-08-01

Journal:PROTEIN EXPRESSION AND PURIFICATION

Included Journals:SCIE、PubMed

Volume:90

Issue:2

Page Number:178-185

ISSN No.:1046-5928

Key Words:Pichia pastoris; Protein purification; Secretory expression; Staphylococcal Protein A

Abstract:Staphylococcal Protein A (SPA), a cell wall protein of Staphylococcus aureus, is in high demand because of its ability to bind immunoglobulins. Much of the SPA that we use today is recombinant SPA (rSPA), which is produced in Escherichia coli. As rSPA is obtained by expressing SPA as an intracellular protein, its purification is tedious and time consuming. In order to obtain a large amount of highly purified rSPA with relative ease, we expressed SPA as a secretory form in the yeast Pichia pastoris. To increase the expression level of SPA and repress its proteolysis during fermentation, the cell density (OD600), temperature and pH at which SPA expression was induced as well as the induction time were optimized. The final yield of SPA obtained was about 8.8 g per liter of culture, which under the optimized fermentation condition, accounted for 80% of the total protein in the culture supernatant. The expressed SPA was purified from the culture supernatant by DEAE ion-exchange.chromatography (IEC) after the supernatant was subjected to a desalting step. The purified SPA was resolved as a single band by SDS-PAGE and as a single peak by HPLC. Its identity was confirmed by MALDI-TOF MS and western-blot. Moreover, the protein also exhibited excellent affinity for IgG when tested with human IgG. The production and purification of SPA described in this study offers a new method for obtaining high level of SPA in relatively pure form that is suitable for practical application. (C) 2013 Elsevier Inc. All rights reserved.

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