合成人干细胞cDNA在大肠杆菌中的高效表达与纯化  

The high expression and purification of the synthetic human stem cell factor cDNA

作　　者：赵志虎[1] 马清钧[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2004年第2期108-111,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划专项基金(2002AA227022);国家自然科学基金(30200004)

摘　　要：以pBV220为载体,进行了合成可溶型人干细胞因子(SCF)cDNA在大肠杆菌中的温控型的高效表达。SDS-PAGE检测表明,在实验室摇瓶培养中,目的蛋白可占菌体可溶蛋白的40%左右。表达产物复性后,经过凝胶过滤、离子交换层析,得到了电泳纯的重组rhSCF,经测定,纯化的rhSCF相对分子质量为19000,氨基端15个氨基酸的序列与天然可溶形式的hSCF成熟分子的序列完全一致。The high-level expression of the cDNA for soluble human stem cell factor (hSCF) in Escherichia coli and the purification of the recombinant hSCF product were reported here. After the sequencing confirmation, the synthetic soluble form hSCF cDNA was inserted into pBV220 which was then transformed E.coli DH5α for expression. The SDS-PAGE analysis of total cell extract showed that the hSCF was expressed at a high level in E.coli and the hSCF protein consisted about 40% of the total soluble protein. After oxidation and refolding, the crude extract was applied onto anion-exchange chromatography and gel filtration chromatography. With these procedures, a homogeneous rhSCF was obtained, which had a molecular weight of 19.0 kD and the same amino terminus sequence to that of the natural hSCF.

关 键 词：人干细胞因子 CDNA 大肠杆菌 表达 纯化 蛋白 

分 类 号：Q786[生物学—分子生物学] Q51