Met-Lys-双C肽人胰岛素原基因的构建表达及分离纯化

应用 Ｐ Ｃ Ｒ 定点突变方法构建编码 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原基因，并在大肠杆菌中以包含体方式获得表达 表达产物经还原、重组、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 分离纯化，获得 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原，经胰蛋白酶与羧肽酶 Ｂ的酶解， Ｒｅｓｏｕｒｃｅ Ｔ Ｍ Ｑ 阴离子交换柱层析分离制备得人胰岛素，其放免活性、受体结合活性均与猪胰岛素相同 

Construction of Met Lys Double C Peptide Human Proinsulin Gene and Its Expression and Characterization

A fusion gene encoding Met Lys double C peptide human proinsulin was constructed with PCR The mutation was confirmed by DNA sequencing,and it was expressed in E coli system in inclusion body form The temperature inducible promoter was employed for induction in a short time After reducing and refolding,expressed recombinant products could be easily purified by Sephadex G 75 chromatography The Met Lys double C peptide human proinsulin analogue could be changed into human insulin with the treatment of trypsin and carboxypeptidase B After separation with anion exchange chromatography Resource TM Q column,human insulin could be obtained Polyacrylamide gel electrophoresis analysis showed the purified human proinsulin and human insulin Its radioimmuno activity and receptor binding activity were almost the same as those of porcine insulin Met Lys double C peptide human proinsulin converted to human insulin with correct disulfide bonds This strongly suggested the existence of very flexible conformation of C peptide The addition of Lysine between Met(initiator)and Phe(B 1)could easily produce human insulin with N terminal Phe (B 1) from the human proinsulin analogue in E coli system