突变型人膜联蛋白V的重组表达

Recombinant Expression of Mutant Human Annexin V

目的 将人膜联蛋白V基因突变后转化入毕赤酵母重组表达，从培养液中纯化出具有内在金属螫合位点的活性突变型人膜联蛋白V。方法 应用特异引物将天然人膜联蛋白V基因的5’和3’端进行突变改造，将突变型膜联蛋白V基因插入到pPIC9K质粒并进行基因测序鉴定。将正确连接的表达质粒线性化后用电穿孔法转化到毕赤酵母GS115中。通过MD平板筛选转化子、BMGY培养基培养、甲醇诱导表达目的蛋白质。培养物经过离心获取上清液，用SDS-PAGE和银染分析蛋白质的表达情况，通过外露磷脂酰丝氨酸的红细胞和异硫氰酸荧光素标记膜联蛋白V测定上清液中突变型人膜联蛋白V的结合活性。结果 天然人膜联蛋白V基因5’端融合GCAGCGGCTGCGGCCAT编码序列，3’端946-948位TGT突变为AGC。毕赤酵母转化子分泌产生相对分子质量为36000的蛋白质，其半数抑制浓度为4nmol/L。结论 利用毕赤酵母系统表达出高活性的、具有内在金属螫合位点的突变型人膜联蛋白V。

Objective To mutate human annexin V gene and transform it to Pichia Pastoris for mutant human annexin V expression, so as to be purified as active annexin V with endogenous metal chelating site. Methods The 5＇ and 3＇ end of native annexin V gene were mutated by specific primers. The mutant annexin V gene was inserted into pPIC9K and sequenced. The correct plasmid was linearized and transformed into Pichia Pastoris strain GS115 by electroporation. The transformants were selected from MD plates and cultured in BMGY medium and induced with reathanol. The culture was centrifuged and the supernatant was analyzed by SDS-PAGE and silver staining. The binding activity of mutant human annexin V from culture supernatant was determined with phosphatidylserine exposed erythrocytes and fluorescein isothiocyanate-annexin V. Results The 5＇ end of native human annexin V gene was fused with GCAGGCGGCTGCGGCCAT coding sequence and 3＇ end 946-948 site TGT was mutated to AGC. Pichia Pastoris transformants secreted proteins of relative molecular mass 36 000 48 h after methanol induction. The concentration of this protein that inhibited 50% of the binding of fluorescein-annexin V was 4nmol/L Conclusion Highly-active recombinant mutant human annexin V with endogenous metal-chelating sites can be expressed in Pichia Pastoris system.