GX1-rmhTNFα工程菌发酵及表达产物的纯化

Fermentation of recombinant E.coli expressing GX1-rmhTNFα and purification of expressed product

目的全面检定GX1-rmhTNFα工程菌,并建立稳定的发酵及纯化工艺。方法对GX1-rmhTNFα工程菌进行全面检定,挑取目的蛋白表达水平高的菌种扩大培养;采用5 L发酵罐,32℃培养工程菌至菌体A600值达4～6时,42℃诱导约4 h后收菌,发酵过程控制pH值在7.2,溶氧在30%左右,采用梯度流加的方法补料,连续发酵3批,验证发酵工艺的稳定性;发酵产物经硫酸铵分段盐析及透析处理后,经SP-Sepharose FF及Q-Sepharose FF层析纯化,收集洗脱峰进行纯度、浓度、活性及Western blot分析。结果工程菌经全面检定,证实构建正确,目的蛋白表达量不低于10%,菌种稳定性较好,呈典型的大肠杆菌特征,适合建立工程菌菌库;连续发酵3批,湿菌收量均不低于200 g,目的蛋白表达水平均在10%以上;目的蛋白经柱层析纯化,最终纯化产物蛋白含量为0.901 mg/ml,活性为5.97×108IU/ml,纯度可达95%以上,Western blot分析显示,目的蛋白可与鼠抗人TNF单抗特异性结合。结论初步建立了稳定可行的GX1-rmhTNFα融合蛋白的发酵及纯化工艺。

Objective To perform overall control tests on recombinant E.coli expressing GX1-rmhTNFα and develop a stable procedure for fermentation and purification.Methods The recombinant E.coli expressing GX1-rmhTNFα was subjected to overall control tests,and the ones in which target protein was highly expressed were selected for scale-up culture in 5 L fermenter at 32℃ until the A600 value reached 4 ～ 6,then induced at 42℃ for about 4 h and harvested.The pH value during fermentation was controlled at 7.2,while the DO at about 30%,and the supplements were added by gradient flow feeding.The fermentation procedure was verified for stability by fermentation of three consecutive batches of recombinant E.coli strain.The fermentation product was subjected to fractional salting out with ammonium sulfate and dialysis,then purified by SP-Sepharose FF and Q-Sepharose FF chromatography.The elution peaks were collected,determined for purity,concentration and activity,and analyzed by Western blot.Results Overall control tests proved that the recombinant E.coli was constructed correctly,in which the expression level of target protein was not less than 10%.The bacterial seeds showed high stability and typical character of E.coli,which was suitable for establishment of recombinant E.coli seed bank.The harvests of wet bacteria of three consecutive batches of fermentation product were not less than 200 g,in all of which the expression levels of target protein were more than 10%.The protein content,activity and purity of purification product were 0.901 mg / ml,5.97 × 10^8 IU / ml and more than 95% respectively.Western blot showed specific binding of target protein to mouse anti-human TNF monoclonal antibody.Conclusion A stable and feasible procedure for fermentation of recombinant E.coli and purification of GX1-rmhTNFα fusion protein was preliminarily developed.