重组人可溶性TRAIL在毕赤酵母中表达与纯化及其抗肿瘤细胞活性

Expression and purification of human soluble TRAIL in Pichia pastoris and its anti-tumor activity in vitro

目的研究重组人可溶性TRAIL(sTRAIL)蛋白的表达和纯化,以及对HepG2细胞增殖与凋亡的影响。方法将人sTRAIL基因插入毕赤酵母表达载体pPIC9中,构建甲醇酵母分泌型表达载体pPIC9-sTRAIL,电击转化至GS115(his4),MD平板筛选出阳性菌株。对工程菌株发酵培养基成分、pH、甲醇浓度、诱导表达时间等条件进行优化,并初步分析重组sTRAIL蛋白的体外抗肿瘤活性。结果工程菌株在BMMY培养基(pH6.0)、1%甲醇条件下诱导表达48h目的蛋白浓度最高可达(58.7±2.4)mg/L。重组人sTRAIL蛋白能抑制HepG2细胞的增殖、诱导HepG2细胞的凋亡。结论优化了人sTRAIL的表达和纯化工艺条件,所生产的sTRAIL可用于抗肝癌新药的开发。

Purpose To investigate human soluble TRAIL （sTRAIL） protein expression and purification and its potential anti-tumor activity on hepatocellular carcinoma （HepG2）.Methods Soluble TRAIL gene ligated with expression vector pPIC9 was transfected into GS115 （his4） and the recombination strain expressing sTRAIL was screened by MD plate.The effects of different media,methanol inducement period,methanol concentration,and pH were investigated and optimized using shaking flask.The anti-tumor activity of sTRAIL with HepG2 cells was analyzed after purification.Results The highest expression of sTRAIL was obtained at pH 6.0,1% methanol in BMMY medium,with the concentration of （58.7±2.4） mg/L at 48 h.Recombinant sTRAIL protein could induce HepG2 cells apoptosis and inhibit HepG2 cells proliferation effectively.Conclusion The optimized condition of human sTRAIL expression and purification was developed and the obtained recombinant sTRAIL protein may be a promising therapeutic agent for hepatocellular carcinoma.