The Preparation Of Recombinant Fusion Protein RMBP-NAP

Our research group had constructed and screened the Genetic engineering bacteria E. coli TB1(pMAL-c2x-napA) which can express the fusion protein MBP and NAP, the pharmacodynamic studies showed that the recombinant fusion protein rMBP-NAP played an antitumor role in the mice model by enhancing secretion of Th1-type cytokines. In order to optimizing the experimental shake flask system for E. coli TB1(pMAL-c2x-napA) in the laboratory and reducing the cost to build the small scale fermentation process and providing parameters for pilot plant experiment, the preparation of recombinant fusion protein rMBP-NAP was studied which including the fermentation medium, shake flask process and laboratory scale test in a10L fermenter.Methods(1) Optimization of the medium and fermentation condition in a250mL shake flaskThe fermentation was carried out in a250mL flask, incubated in a shaking table whose speed was kept at120~150rpm. The conditions for shake flask fermentation include medium, spawn age, inoculum size, volume of medium, induction time, inductor concentration, initial pH of the medium, and induction temperature. Only one parameter was varied at a time during the optimization process.(2) Comparison of condition between optimized and not optimizedAfter we finished the optimization of the fermentation, we tested the optimized condition and not optimized condition for the fermentation process at the same time. the expression and yield of rMBP-NAP the terminal cell concentration of E. coli TB1(OD600) were compared.(3) Laboratory scale test in a10L fermenterThe laboratory scale test was carried out in a10L fermenter (Zhen Jiang, Dong Fang, Biotech), which was based on the optimized condition (dada above on). The curve of the cell growth kinetics and the curve of rMBP-NAP expression were obtained by comparing the terminal cell concentration(OD600) and expression of rMBP-NAP at the end of fermentation process.Results(1) The optimal culture conditions for the production of the recombinant protein in shake flask were as follows: M9medium (with3%yeast extract powder added), the spawn age of19h，the inoculum size of6%，the initial pH of the medium6.6, temperature at37°C and0.7mMIPTG inducted21h in a50mL/250mL shake flask. By comparison, the yield of rMBP-NAP was improved significantly, which was from59mg/L increased to592mg/L, nearly increased by10times when the optimized fermentation conditions was used in shake flask.(2) The laboratory scale test in a10L fermenter could get30g wet cell per liter fermentation broth with the concentration of the soluble protein of1738mg/L, and the soluble rMBP-NAP accounted for10.4%of the crude extract protein.Conclusion(1) The optimal culture conditions for the production of the recombinant protein in shake flask were selected out. The yield of the fusion protein was increased by9.04%after optimization. The study could provide an optimal condition for the shake flask cultivation and increase the expression and the productivity of rMBP-NAP.(2) The result rooted in the10L fermenter could provide some proofs for the further study and lay a foundation for scaling up the fermentation process to pilot plant experiment.