Li Lin, Qian Dongmeng, Shao Guangcan, Yan Zhiyong, Li Ronggui, Hua Xiaomin, Song Xuxia, Wang Bin
Department of Microbiology, Qingdao University Medical College, Qingdao 266071, People's Republic of China.
Department of Biology, Qingdao University College of Life Science, Qingdao 266071, People's Republic of China.
Protein Expr Purif. 2014 Sep;101:99-105. doi: 10.1016/j.pep.2014.06.008. Epub 2014 Jun 20.
M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.
M-IL-2（（88）精氨酸，（125）丙氨酸）是一种融合蛋白，由与突变型人白细胞介素2（（88）精氨酸，（125）丙氨酸）基因连接的蜂毒肽组成。在本研究中，我们构建了M-IL-2（（88）精氨酸，（125）丙氨酸）在毕赤酵母中的表达系统：GS115/pPICZα A/M-IL-2（（88）精氨酸，（125）丙氨酸），并实现了该融合蛋白的高水平表达。融合蛋白M-IL-2（（88）精氨酸,（125）丙氨酸）的最大产量达到814.5mg/L，高于大肠杆菌表达系统。通过硫酸铵分级沉淀、透析和镍离子亲和层析对融合蛋白进行纯化。融合蛋白的分子量约为26kDa，与理论值相符。通过蛋白质免疫印迹检测表明M-IL-2（（88）精氨酸，（125）丙氨酸）具有较强的抗原特异性。生物活性检测结果表明，该融合蛋白在体外可直接抑制人卵巢癌SKOV3细胞和Hela细胞的生长。本研究为以毕赤酵母作为表达宿主大规模生产具有生物活性的M-IL-2（（88）精氨酸，（125）丙氨酸）提供了一种替代策略，并为其临床应用铺平了道路。
