6个氨基酸小C肽人胰岛素原类似物基因的构建和表达

Construction and Expression of the Gene for“Mini-C”Analog of Human Proinsuli

用片段置换法，从在Ｃ肽两端具有酶切位点的双突人胰岛素原基因中，将Ｃ肽基因替换成含Ａｒｇ－Ａｒｇ－Ｇｌｙ－Ｓｅｒ－Ａｒｇ－Ｌｙｓ６个氨基酸小Ｃ肽基因。将这个小Ｃ肽人胰岛素原类似物基因（ＢＣ＇Ａ）重组到具有Ｔａｃ启动子的质粒中，并与部分牛凝乳酶原基因融合，在Ｅ．ｃｏｌｉ中得到了高效表达。表达的ＢＣ＇Ａ 融合蛋白占菌体总蛋白的１６％。表达产物以包含体形式存在，经ＣＮＢｒ裂解及磺酸化，再经复性后，具有对胰岛素的放射免疫活性。

The study of the expression and processing of insulin precursors in yeast shows that changing the length of the C peptide region of insulin to 6 or 2 amino acid residues increased the expression level of insulin like peptide in yeast(Thim et al).For studying expression of insulin precursors in E. coli,one gene has been con-structed by recombinant DNA technology. This gene codes for an analog of human proinsulin in which the 35- amino acid connecting peptide is replaced by a mini-C peptide of six rexidues:Arg-Arg-Gly-Ser-Arg-Lys. This gene was cloned and highly expressed as a fusion protein in E. coli. The expression level accounted for 16%of total bacterial protein.This expression product shows radioimmuno activi-ties of insulin after cleavage with CNBr,sufitolysis and refolding treatment.The above result may be an efficient route to the production of human insulin,and also useful for investigations of protein folding and for the interaction between A and B chain of the insulin molecule.