Production Technique Of Recombinant Staphylococcal Protein A In Pichia Pastoris

Staphylococcal protein A (SPA) is a cell wall protein of Staphylococcus aureus. Due to its high affinity to immumoglobuins, SPA has been widely used in antibody purification, detection, pathogen rapid diagnosis, immunology and other biological research. Currently, most of SPA is recombinant SPA (rSPA), which is expressed in cells of E. coli through genetic engineering. The complex purification of the intracellular rSPA made a low yield and high cost. So in this study, Pichia pastoris was chosen for rSPA production. The high-cell density fermentation and the purification were investigated to establish a new procedure for rSPA production.Firstly, before fermentation, expression conditions of rSPA were estabilished in shake-flask culture with P. pastoris rGS115-spa, which contains the gene of rSPA, including inducing temperature, pH, inducer concentration, inducing duration and so on. It was a preparation for the following fermentation procedure.Secondly, based on the previous shake-flask work, the inducing cell density, temperature, pH and duration were further optimized in a5L fermenter. At last, the optimal condition for rSPA fermentation was22℃, pH at3.0and kept for60-80h after the addition of methanol at OD600300to350. The highest production was about8.8g/L, account for over80%of total protein in the fermentation supernantant, and the expression level was elevated quite a lot than that in E. coli.Then, the fermentation supernatant was obtained for further purification investigation. As a secretory protein, without cell destruction and DNA precipitation, the pigments removal and degradation separation were key problems for rSPA purification. The pigments needed to be removed before chromatographic purification to repress the irreversible combination to chromatographic media. To remove the pigments in the fermentation supernatant, several methads were tried. The crude supernatant was first precipitated by (NH4)2SO4or ethanol, and then followed with the adsorption by resin-based actived carbon spheres. The crude fermentation supernatant was also tried to load on desalting column directly. Finally, the desalting column turned out to be the most covinient for pigments removal, with highest protein recovery and best pigment removal effect. The aliquot, eluted from desalting column, was further purified by DEAE IEC, which could remove almost all the degraded fragments. The final product showed a single peak in HPLC analysis, and the recovery of the purification was79.4%.At last, the purified product 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 result of this study offers a new method for rSPA high-level expression and purification. Compare to the original production procedures, the yield was elevated and the cost was reduced with the high cell density fermentation and simple purification process.