人胰岛素B27K-DTrI前体在毕氏酵母中表达条件的优化

Optimization of Expression Condition of Recombinant Human Insulin B27K-DTrI Precursor in P.pastoris

目的优化甲醇酵母表达系统,提高B27K-DTrI前体表达量。方法用不同浓度的G418 YPD平板、联合Real-time PCR筛选高拷贝菌株。优化高拷贝菌株的最佳起始诱导吸光度A、诱导时间、诱导剂浓度等表达条件。结果筛选到高拷贝菌株S3菌,最佳诱导条件为诱导起始A600约6.0,甲醇诱导浓度0.75%,诱导时间72 h,目的蛋白表达量118 mg/L。结论通过条件优化实验,为人胰岛素B27K的产业化提供了可靠的实验基础。

Objective To enhance the expression of monomeric insulin B27K-DTrI precursor by optimizing the expression condition in P pastoris. Methods The recombinants were screened on plates with the different concentrations of G418. And then the copies of target gene were detected by Real-time PCR. The expression condition of high-copy strain of B27K-DTrI precursor was optimized, including the initial A, induction time, concentration of inductor, and so on. Results The recombinant S3 with high copies of target gene was obtained. The optimal induction condition was initial A600 6.0, 0.75% methanol induction for 72 h. Under this condition, the expression yield of B27K- DTrI precursor was 118 mg/L. Conclusion The optimization experiment can be a reliable basis for the industrialization of human insulin B27K.