B22Asn人胰岛素突变体的研究

通过ＤＮＡ定点突变法将人胰岛素Ｂ２２Ａｒｇ改造成Ｂ２２Ａｓｎ，去除其一级结构中碱性蛋白酶位点，以期提高胰岛素在体内的稳定性。突变体基因克隆到表达载体ｐＢＶ２２０中，在Ｅ．ｃｏｌｉＤＨ５α中进行表达。分离纯化后的表达产物经胰蛋白酶和羧肽酶Ｂ联合作用获得重组Ｂ２２Ａｓｎ－人胰岛素．该突变胰岛素具有抗胰蛋白酶水解能力，但是其与受体的结合能力只有标准猪胰岛素的１２．４％．胰岛素结构中Ｂ２２Ａｒｇ可能在胰岛素与其受体结合过程中起重要作用。

Studies on B22Asn-Insulin expressed in E. coli

To investigate the stability of insulin and the interaction between insulin and its receptor,the original codon of B22 Arg was mutated to that of Asn through site-directed mutagenesis on proinsulin cDNA. After cloned into an expression vector PBV220,the mutated cDNA was introduced into E. coli strain,DH5α. The B22Asn-proinsulin was expressed as inclusion bodies at a level of 12% of total cell proteins. After sonication,dissolving,refolding and purification on Sephadex G-50,B22Asn-proinsulin was achieved. Under the treatment of trypsin and carboxypeptidase, B22Asn-proinsulin could be processed to B22Asn-insulin,which showed high resistance to trypsin digestion. But B22Asn-insulin had just 12. 4% binding activity with the receptor and 22. 0% RIA activity of those of porcine insulin. These results suggest that B22 Arg in the β-turn region of insulin plays an important role in the interactions between insulin and its receptor.