Research On Generic Fermentation Technology For Heterologous Proteins Expressed By Recombinant E. Coli

Because of the thoroughly research background, low cost, easy operation and other characteristics, Escherichia coli is still the most widely used expression host in research of heterologous proteins. With the in-depth study of E. coli fermentation process, factors that affect fermentation conditions, such as composition of culture medium, accumulation of metabolic inhibitor, fed-batch control strategies and so on, have been revealed. Because of the consistency of the host, a generic high density fermentation technology suitable for E. coli expression system can be summarized through the study. It simplifies the repeatable optimization of fermentation parameters for every protein expressed by E. coli, so the rapid optimization of fermentation process and the reduction of costs can come true. In this paper, we summarized the generic change of fermentation parameters based on the previous technical experience and the fermentation research of recombinant human growth hormone ?rhGH? and heat-labile enterotoxin ?LT?, aiming to make recombinant protein fermentation technology simple and general.The paper mainly studied three kinds of feeding methods on rhGH expression and fermentation process:the pH-stat flow, variable-speed feeding combined with constant-speed, variable-speed feeding combined with pH-stat. The biomass concentration, respiratory activity, protein content, CO₂ emissions and ammonium ion concentration and other parameters were investigated. When using feeding strategy of variable-speed feeding combined with pH-stat, the cell respiratory activity, protein expression and cell growth reached the optimal balance. And successly improved the expression level of rhGH upto 678 mg/L. For expression of other proteins, LT?ONC and other heterologous protein, we used the variable-speed feeding combined with pH-stat fed-batch strategy to maintain the expression levels in 20% and above.Because of the complexity of inclusion body purification process, soluble expression of protein has extensive research value and application prospect. We have done some research on LT soluble expression technology, and finally established the soluble protein fermentation condition:3 g/L glucose; induced in 5.5 h by 0.15 mM IPTG; 1 g/L sucrose and 150 ?g/mL Kanamycin were added after induction. After fed-batch fermentation in 5L tank, soluble protein levels increased to 6.5% from under 2%. We simultaneously established purification technology of inclusion body by affinity chromatography and gel chromatography. Finally, the purity of target protein was 92.1% and the yield of protein purification was 9.0%.