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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 establish a high-density fermentation process of recombinant human bone morphogenetic protein-7 (rhBMP-7). Methods The strain BL21 / pBV221-rhBMP-7 was cultured in shake flask and fermenter. The effects of different media, acetic acid concentration, pH value and induction time on the growth of the engineered bacteria and the expression of the target protein were observed. The engineered bacteria were cultured under the optimized fermentation conditions. When the A600 value reached 100, the temperature was raised at 42 ℃, and the expressed product was purified. Results There was no significant difference in the expression of target proteins among the engineered bacteria cultivated in the fermentation medium and the LB medium. Acetic acid significantly inhibited the growth of the cells and the expression of the target protein. The optimum pH for the growth of the cells and the expression of the target protein were 6.8 7.6; the best induction time is 3h. After 3 hours of induction, the target protein could reach 34.9% of the total bacterial protein and the final cell A600 value reached 139.5. The purity of the purified target protein was more than 95%. Conclusion The high-density fermentation of rhBMP-7 engineered bacteria has been initially established.