title: High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein.
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.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Hao
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Hongde
authors.initials: H
authors.lastname: He
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Du
authors.forename: Lingyun
authors.initials: L
authors.lastname: Jia
mesh_terms: D002852:Chromatography, Ion Exchange; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
keywords: Pichia pastoris; Protein purification; Secretory expression; Staphylococcal Protein A