毕赤酵母工程菌表达人白细胞介素——2突变体发酵条件的优化

Optimization of Conditions to Stabilize and Maximize Pichia pastoris Fermentation of Recombinant Mutant Interleukin2

巴斯德毕赤酵母(Pichia pastoris)工程菌(KM71/pPIC9K-MvhIL-2,本室构建)能分泌表达三突变体人白细胞介素-2(MvhIL-2),突变位点分别为125 Cys→Ala;18leu→Met;19leu→Ser.通过对其发酵条件的研究,探索重组人白细胞介素-2突变体工程菌KM71/pPIC9K-IL-2的生长及产物表达规律,通过3因素(即:发酵液起始pH值、甲醇诱导浓度、诱导时间)从3个不同水平上进行正交试验,聚丙烯酰胺凝胶电泳分析,得出摇瓶发酵表达MvhIL-2的最佳条件:发酵液初始pH 6.0,甲醇诱导浓度为0.5%,诱导72 h,最终使目的产物达到菌体总蛋白的30%以上,表达的MvhIL-2考马斯亮蓝染色条带最明显,为其进一步上发酵罐,大量生产重组人白细胞介素-2奠定了基础.

The engineering strain of Pichia pastoris, KM71/pPIC9K-MvhIL-2, which was constructed by our lab, can express secretory human mutant interleukin-2 （the codon for cysteine-125 of human IL-2 with alanine, the codon for leucine-18 with methionine and the codon for leucine-19 with serine）. This paper reports a study of the fermentation conditions of KM71/pPIC9K-MvhIL-2 to find the pattern of the growth and expression of the target protein. The expression conditions of recombinant human mutant interleukin- 2 were optimized via orthogonal test of three factors （initial medium pH, methanol concentration and the induction duration） at three levels. The target protein was detected and analyzed with SDS-PAGE. The optimum induction conditions for mutant interleukin-2 expression were： an initial pH of 6.0, a final methanol concentration of 0. 5 %, and 72 hours for induction. Under these conditions, the expressed mutant IL- 2 was secreted into the fermentation broth and a yield of 30% was achieved. A distinct band stained with Coomassie Brilliant Blue for the expression of MvhIL-2 was obtained.