The Expression Of Foreign Protein In Recombinant Strains High Cell Density Culture

Escherichia coli and Pichia pastoris are broadly used as the host strains in genetic engineering and molecular biology. Many important and valuable protein and polypeptide have been expressed in E. coli or P. pastoris. The combination of recombinant DNA technology and large-scale culture process has enabled recombinant proteins to be produced in massive quantities.In this thesis, human B lymphocyte stimulator (hsBLyS) and the GST fussion protein Glycerol dehydratase medium subunit-GST (dhaB1-GST) expressed in the Escherichia coli and recombinant Rhizopus arrhizus lipase expressed in the Pichia pastoris expression system were being systematically and thoroughly investigated.The process of expression of hBLyS (human B Lymphocyte cell stimulator) gene under the control of T7promoter in Escherichia coli was investigated. Lactose was used as an inducer instead of the expensive nonmetabolizable analog of lactose, IPTG. It was found that lactose could induce foreign protein expression and enhance cell growth during the induction period. Through proper optimization of culture and induction conditions, a high cell density OD600=57and an expression level about21%of total cellular protein was achieved, which was very close to the level (26%) when using IPTG as inducer in the shake flask.The Effect of different induction condition:the type of inducer, the concentration of the inducer and the induction temperature, on dhaB1-GST fussion protein soluble expression in Escherichia coli was investigated. Under the optimum condition (22mM lactose as the inducer,30℃induction), the expression level of the dhaBl-GST fussion protein was about14%of total cellular protein, and the soluble expression level was about13%of total soluble protein.In this thesis, the extracellularly active production of recombinant Rhizopus arrhizus lipase (RAL) was carried out by the expression the genes encoding the mature region (mRAL) and the m-RAL having the prosequence (Pro-RAL) in Pichia pastoris. Two transformed Pichia pastoris clones containing the m-RAL and Pro-RAL genes were separately selected for the production of recombinant enzymes. In shaker flask culture, the recombinant lipase activity of both strains were achieved about1.5U ml-1. After optimization of the culture and methanol induction condition, in a5L fermentor a very high lipase activity (Pro-RAL) about88.2U ml-1was achieved after42h of cultivation by using on-line methanol analyzer for control of methanol feeding.