培养条件对重组毕赤酵母高密度表达猪胰岛素前体的影响

Culture Conditions Affecting High Level Expression of Porcine Insulin Precursor(PIP) by Recombinant Pichia pastoris in High Cell Density Fermentation

研究了基因工程菌P ich ia p astotis高密度、高表达的培养条件。在全合成摇瓶培养基的基础上,考察了诱导过程中甲醇浓度、温度、pH、(NH4)2SO4及磷酸盐浓度等因素对猪胰岛素前体(P IP)表达的影响。研究表明:甲醇的最佳诱导浓度为6 g/L,过高或过低的甲醇浓度都不利于菌体生长和蛋白表达;在菌体生长和蛋白表达阶段,pH应分别控制在5.5和4.0;诱导阶段,培养温度下降到28°C,可以提高重组蛋白质的表达量。上述结果用于15 L全自动发酵罐实验,P IP表达水平达到了1.69 g/L,是摇瓶结果的17倍。

The cultivation conditions for porcine insulin precursor （PIP） production by recombinant Pichia pastoris were optimized in shake flasks with synthetic fermentation media. The influences of pH, temperature, the concentrations of methanol, （NH4）2SO4, and phosphate on the expression of PIP and the cell growth were investigated. The optimum concentration of methanol is 6 g/L. The optimal pH value is 5.5 at cell growth phase and 4. 0 in expression phase. Meanwhile, reducing the culture temperature to 28 ℃ in induction phase can increase the PIP expression level. The above results are successfully applied to a 15 L fermenter, where the PIP concentration reach 1.69 g/L, which is 17 times as high as that in the shake flasks.