Expression Of Recombinant Protein A And Fermentation Process Optimization Of Recombinant Cells

Staphylococcus aureus protein A (SPA) which can specifically bind a variety of mammalian antibodies has a wide range of applications in antibody analysis, bioseparation and medicine. As protein A is a cell wall binding protein of pathogenic staphylococcus aureus, producing protein A by large-scale cultivation of staphylococcus aureus is risk and the purification process is also sophisticated.The research aims to produce recombinant protein A by genetic engineering technology in a safe and simple way, covering the construction of genetical engineering strains for expressing protein A and the optimization of culture conditions in shake flask and fermentor.First, prokaryotic expression system was selected. E.coli, pET series plasmid, and different lengths of protein A coding genes containing antibody binding domain were used as expression host, expression vector and target genes, respectively. Four different lengths of intracellular protein A with molecular weights were obtained. The results showed that spa4 had the maximum expression quantity and a good IgG binding activity. This strain was selected for further optimization of the culture and induction conditions in shake flask and fermentor. The effects of inducer concentration and adding time on bacteria growth and protein A expression were preliminarily studied in shake flask. The results showed that the engineering bacteria gradually increased with the delay of inducer added in 1 h to 6 h cell culture period, and the expression quantity per mass of bacteria did not change significantly. Besides, isopropyl (3-D-thiogalactopyranoside (IPTG) and lactose could effectively induce the expression of protein A, and lactose had the weaker inhibitory effect on bacteria growth. In 5 L fermentor, feeding rate was optimized. The optimal conditions for fermentation were obtained:the feeding rate of medium contained 300 g/L glucose,50 g/L yeast extract was calculated as theoretical specific growth rate of 0.1 h-1. IPTG was added to the concentration of 0.5 mmol/L when OD600nm was up to 60. The cell density of OD600nm in the final fermentation broth could reach more than 70, and the yield of recombinant protein A could be up to 2.6 g/L broth which accounted for about 22% of the total intracellular protein.In order to simplify the purification process of recombinant protein A and reduce its cost, eukaryotic expression system was studied further to realize the excretion of recombinant protein A. Genetic engineering yeast that could excrete recombinant protein A was successfully constructed by using Pichia pastoris GS115, pPIC9K and protein A coding genes containing only antibody binding domain as expression host, vector and target gene, respectively. In shake flask, effects of medium composition, concentration of methanol as the inducer and induction time on the expression of protein A were investigated. The results turned out that the optimal conditions were using BMGY/BMMY as medium and adding methanol to 0.5% every 24 h during the induction time of 84 h. The recombinant protein A had a good IgG binding activity.In this paper prokaryotic and eukaryotic expression system of recombinant protein A was established and culture and induction conditions were optimized. The results laid the foundation for large-scale production of recombinant protein A.