一种重组抗胰蛋白酶单体人胰岛素突变体的设计、制备和鉴定

用缺口双链DNA的定向突变方法分别将胰岛素前体中B链第 2 2、2 8、2 9和 3 0位改变为Asp、Lys、Pro和Lys,酵母分泌表达的前体经胰蛋白酶直接酶切 ,得到重组 [B2 2Asp、B2 8Lys、B2 9Pro、B3 0Lys]人胰岛素。它与受体的结合能力约为猪胰岛素的 6% ,而体内生物活力保留 5 0 %。通过FPLC分子筛测定其自身结合能力 ,在生理条件下浓度达 10 -4mol/L时它以单体形式存在。作为可抗胰蛋白酶酶解的单体胰岛素类似物 ,它可能具有一定的应用前景

A Recombinant Monomeric Human Insulin Mutant with Resistance to Trypsin: Desig n, Preparation and Characterization

By using a gapped duplex DNA method, the four amino a cids on the B chain of insulin, i.e. B22Arg, B28Pro, B29Lys and B30Thr, were mut agenized simultaneously into B22Asp, B28Lys, B29Pro and B30Lys, respectively. Th e recombinant [B22Asp?B28Lys?B29Pro?B30Lys]human insulin was obtained simpl y by the treatment with trypsin of the precursor that was expressed in yeast. Th is human insulin analog had only 6% of receptor binding activity as that of the porcine insulin, but retained 50% of in vivo biological activity, compared w ith the latter. The self association ability of the mutant measured through FPL C gel filtration chromatography showed that it was in monomeric state. As a mono meric insulin analog with resistance to the trypsin digestion, this compound may be promising in practical application.