人肝再生增强因子在毕赤酵母中的表达、纯化和生物学活性的研究  

Expression and Purification of Human Augmenter of Liver Regeneration in Pichia pastoris and Evaluation of its Bioactivity in Vitro

作　　者：陈俊杰[1] 孔祥平 佟明华 李茹冰 杨联萍 游松[2] 

机构地区：[1]解放军458医院全军肝病中心,广州510600 [2]沈阳药科大学制药工程学院,沈阳110016

出　　处：《中国生物工程杂志》2007年第6期22-26,共5页China Biotechnology

基　　金：广东省自然科学基金(04103063)

摘　　要：肝再生增强因子(ALR)是一类胞源性肝细胞生长因子。为在毕赤酵母中分泌表达人肝再生增强因子(rhALR),以色谱法分离纯化后进行体外活性研究,构建表达载体pPICZαA-ALR,经电穿孔转入毕赤酵母中,用0.5%甲醇诱导表达;重组酵母培养上清经SDS-PAGE电泳和western blot鉴定后表明,rhALR以分子量为30kDa的二聚体为主;定量分析结果表明,重组酵母培养上清中rhALR约占总蛋白的66%,表达量约为40mg/L;经DEAE柱和G75柱纯化后,获得的rhALR纯度大于95%,得率为52%;体外生物学活性实验表明,rhALR能明显促进HepG2、SMMC-7721和NIH-3T3细胞的增殖。Augmenter of liver regeneration （ALR） is a kind of hepatocyte growth factor . To study the expression, purification and bioactivity of human augmenter of liver regeneration （rhALR） in Pichia pastoris, the expression plasmid pPICZαA- ALR was constructed and transformed into P. pastoris by the method of electroporation transformation. Induced with 0.5% methanol, the 30 kDa protein in the culture supernatant of recombinant P. pastoris was confirmed to be rhALR by SDS-PAGE and Western blot. Quantitative analysis showed that the target protein was in a level of 66% of the total protein of the culture supernatant, with a yield of 40mg/L. We had performed DEAE anion exchange chromatography two times with excessive and regular adsorption quantity consecutively, and then the rhALR above 95% purity and 52% protein recovery could be obtained by G75 molecular sieve chromatography at last step. The bioactivity assay of the purified product showed that rhALR could stimulate the proliferation of HepG2, SMMC-7721 and NIH-3T3 in vitro.

关 键 词：肝再生增强因子 毕赤酵母 分泌表达 分离纯化 生物学活性 

分 类 号：Q593.1[生物学—生物化学]