Study On High Cell Density Culture Of Recombinant E. Coli For Production Of IgG-binding Protein GST-GB2

Recombinant protein GST-GB2, as one kind of IgG binding protein which has two IgG binding domain,has boad prospects of application in the field of immunology.. Extensive applation of GST-GB2puts forward higher requirements to the large-scale production of GST-GB2and the production in shake flask have not be suitable for the need of application.High cell density culture techniques haved been widely used for the large-scale production of exogenous recomninant protein because it can significantly increase the amount of bacteria in the unit volume thereby enhancing the production of exogenous recombinant protein.In this study, high cell density cultivation of E.coli BL21(PGEX-GB2) has been achieved in a5L fermentor by fed-batch technique.The expression level of GST-GB2in a5L fermentor is also higher than in the shake flask.The results show as follows;1One kind of glucose ammonium salts medium is used for the need of high cell density cultivation of recombinat E.coli. According to the growth characteristics of the strain in the study,the concentration of glucose and ammonium sulfate in the medium is optimized.The strain has the best growth condition when the concertration of glucose is25mg/mL and the concertration of ammonium sulfate is4mg/mL in the medium.For the protection of the glucose in the medium,the glucose is sterilized separately at121℃for20min.2For the protection of the glucose in the medium,the glucose is sterilized separately at121℃for20min.The loss of glucose in this sterilization was only3.5%,it is the best sterilization in this research.3Three kinds of feeding modes-constant rate feeding, pH feedback control feeding, DO feedback control feeding are adapted in the fed-batch process of high cell density cultivation.The cell density(OD6oo) of recombinant E.coli BL21(PGEX-GB2)in three feeding modes are all more than100and the maxium cell density is116.The DO feedback control feeding mode is considered the best feeding mode after the cell yield coefficient Yx/S and average call growth rate are further compared.In this feeding mode,the cell yield coefficient is0.306g/g,and the average cell growth is2.95g/L-h.4In the production phase of GST-GB2,the induction hours of protein is optimized to obtain the maxium yield of GST-GB2. The yield of GST-GB2in the cell of recombinant E.coli reach the highest at6h after induction and the yield is4.12g/L that is2.81times higher than in the shake flask.The activity of GST-GB2from high cell density cultivation is determined by ELISA and the results show that the GST-GB2is the active protein with IgG binding capacity.