脂肪细胞新蛋白My027原核表达、纯化及生物活性研究

目的通过原核表达获得大量脂肪新蛋白质My027,并在体外初步研究其生物活性。方法RT-PCR法扩增出My027片段,插入原核表达载体pGEX-4T-2,构建质粒pGEX-My027。转化BL-21菌,IPTG低温诱导表达融合蛋白,经亲和层析法获得融合蛋白,利用SDS-PAGE、Western印迹及质谱进行鉴定。在还原性谷胱甘肽存在的条件下,将纯化得到的融合蛋白作用于乙二醛酶Ⅰ的底物甲基乙二醛,通过分光光度法检测产物乳酸谷胱甘肽的生成。结果融合蛋白以水溶形式分泌于大肠埃希菌BL-21胞浆中,纯化后的目的蛋白SDS-PAGE、Western印迹及质谱证实高效表达,氨基酸序列正确。结论人脂肪细胞新蛋白质My027在pGEX-4T-2原核表达载体中可获高效表达,所纯化蛋白具有类似于乙二醛酶Ⅰ的作用。

Expression and Purification of a Novel Human Adipocytes Protein My027 and Its Activity Analysis

Objective To obtain abundant novel protein My027 of human adipocytes by eukaryotic expression system,and to identify preliminarily its function in vitro.Methods The full length My 027 cDNA was amplified by RT-PCR from the human adipocytes.The fragment was inserted into the vector pGEX-4T-2 to construct recombinant plasmid p GEX-My027.Following transformation into E.coli BL-21,the fusion protein was expressed under IPTG induction.After purification,the protein My027 was determined by SDS-PAGE,Western blot and Mass Spectometry(MS).The biological activity of the protein My027 was identified by determining its capability of catalyzing methyglyoxal into S-lactoglutathione in vitro.Results High yield and purity of the fusion protein My027 was obtained and confirmed by SDS-PAGE,Western blot,and MS.The purified protein is able to catalyze the methyglyoxal into S-lactoglutathione in vitro,similarly to glyoxalase I.Conclusions The pGEX-4T-2 system produces functional protein My027.The purified protein My027 presents the same function as that of glyoxalase I.