重组人骨形态发生蛋白-7工程菌的高密度发酵

High Density Fermentation of Recombinant E. coli with Human Bone Morphogenetic Protein-7

目的建立重组人骨形态发生蛋白-7(rhBMP-7)工程菌的高密度发酵工艺。方法采用摇瓶及发酵罐培养工程菌BL21/pBV221-rhBMP-7,观察不同培养基、乙酸浓度、pH值、诱导时间等对工程菌菌体生长及目的蛋白表达的影响。在优化的发酵条件下培养工程菌,当菌体A600值达100时,42℃升温诱导,并对表达产物进行纯化。结果发酵培养基与LB培养基培养的工程菌目的蛋白的表达量无明显差异;乙酸可明显抑制菌体生长及目的蛋白表达;最适于菌体生长和目的蛋白表达的pH值分别为6.8和7.6;最佳诱导时间为3h。以优化的发酵条件培养的工程菌诱导3h后,目的蛋白的表达量可达菌体总蛋白的34.9%,最终菌体A600值可达139.5;经纯化的目的蛋白纯度可达95%以上。结论已初步建立了rhBMP-7工程菌的高密度发酵工艺。

Objective To develop a high density fermentation procedure for E.coli expressing recombinant human bone morphogenetic protein-7（rhBMP-7）.Methods Recombinant E.coli BL21 /pBV221-rhBMP-7 was cultured in shake-flask and in fermenter respectively,and the effects of various factors such as medium,acetic acid concentration,pH value and time for induction on growth of recombinant E.coli and expression of target protein were observed.The recombinant E.coli was cultured under optimized condition,and induced at 42℃ when the A600 reached 100.The expressed product was purified.Results The expression levels of target protein in fermentation medium showed no significant difference with that in LB medium.Acetic acid inhibited the growth of recombinant E.coli and expression of target protein significantly.The optimal pH values for growth of recombinant E.coli and for expression of target protein were 6.8 and 7.6 respectively.The optimal time for induction was 3 h.The expression level of target protein 3 h after induction of the recombinant E.coli cultured under optimized condition reached 34.9% of total somatic protein,while the final A600 reached 139.5.The purity of expressed protein was more than 95% after purification.Conclusion A high density fermentation procedure for recombinant E.coli BL21 /pBV221-rhBMP-7 was preliminarily developed.