摘要
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。
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.
