(A11,A12)胰岛素原移位突变体的表征研究

CONSTRUCTION AND CHARACTERIZATION OF MUTANT PROINSULIN WITH SHIFTED INTRA-A CHAIN DISULFIDE-BOND

将(CysA11Ser,SerA12Cys)人胰岛素原移位突变体基因克隆至高效表达质粒pET22b多克隆位点区,构建重组表达质粒pET-A112,并在大肠杆菌E.coli BL21(DE3)pLys中进行了表达.表达产物存在于包涵体中,通过包涵体的分离及二硫键变复性,获得A6-A12二硫键突变型的胰岛素原突变体.SDS-PAGE电泳显示突变型胰岛素原的电泳迁移率与野生型胰岛素原基本相同.质谱检测表明,突变性胰岛素原的分子结构均一、纯度良好,氨基酸组成与理论值基本一致.以野生型人胰岛素原为对照进行的胰岛素受体试验及胰岛素放射免疫试验表明,A6-A12二硫键错接型胰岛素原突变体的受体结合活性和放免活性是野生型胰岛素原的11%与55%.

With PCR technique a mutant proinsulin gene with CysA11Ser and SerA12Cys was constructed to investigate intra A-chain disulfide effect on insulin biological activity. After expression, unfolding and refolding, mutant proinsulin was purified with （A6, A12） intra-A chain disulfide-bond. The mutant proinsulin showed 55% radioimmunoactivity （RIA） and 11% of receptor binding activity of native proinsulin. Mass spectrometry analysis, IEF and amino acid composition assay were carried out to determine mutant proinsulin. Data showed that the A6-A12 intra-A chain disulfide bond was formed, having some negative effect on native proinsulin structure, especially in regard to the stability of insulin receptor binding region.