A链链内二硫键移位突变胰岛素原的构建与表达

采用定点突变技术,将人胰岛素原基因编码框中A11位氨基酸残基与A12位氨基酸残基进行了互换,构建了CysA11Ser,SerA12Cys]人胰岛素原突变基因.在大肠杆菌原核表达系统中对突变体基因进行了表达,并对表达产物进行了变复性研究及初步的分离纯化,获得了具有稳定结构的突变体蛋白质,探讨了突变体A链链内二硫键的形成状况,为进一步深入揭示A链链内二硫键的角色与功能打下了基础.

CONSTRUCTION AND EXPRESSION OF INTRA-A CHAIN DISULFIDE BOND SHIFT MUTANT IN HUMAN PROINSULIN GENE

By using the site-directed mutagenesis technique,encoding-box of A11 amino acid residue and A12 amino acid residue of human proinsulin gene are exchanged and the CysA11Ser,SerA12Cys]-human proinsulin mutant gene is constructed.Finally,the mutant gene is determined by DNA sequencing.CysA11Ser,SerA12Cys]-human proinsulin mutant gene is then expressed in Escherichia coli.The expressed products are treated and purified initially by a denaturation and renaturation process.The mutant protein is stable and is analyzed by SDS-PAGE and amino acid composition assay.DTNB test is also carried out to prove that there is no free-SH in mutant protein.Moreover,the mutant formation conditions in intra-A chain disulfide bond are also tested.All those results above reveal the basic role and function of the mutant in the intra-A chain disulfide bond.