蛋白酪氨酸磷酸酶PTP1B催化活性区的原核表达及活性分析

Abstract

从GenBank获得人PTP1B催化活性区(PTP1Bc)氨基酸序列(1～301aa),通过重叠PCR获得PTP1Bc基因。构建pET-22b(+)/PTP1Bc原核表达载体,转化大肠杆菌BL21(DE3),阳性重组子IPTG诱导表达,Ni柱纯化蛋白。目的蛋白以包涵体的形式表达,表达量占菌体总蛋白30%以上。纯化后,蛋白纯度达95%以上。Western blotting结果表明所得的蛋白可与抗PTP1B抗体发生特异性结合;酶活实验证实复性的蛋白具有一定的磷酸酶活性。PTP1Bc基因的构建、表达纯化及活性分析,为进一步的功能研究奠定了基础。 【英文摘要】 The amino acid sequence (1-301aa) coding the human PTP1B catalytic domain (PTP1Bc) was obtained from the GenBank.The PTP1Bc gene was constructed by overlapping PCR,then was inserted into vector pET-22b(+) and expressed efficiently in E.coli BL21(DE3) under optimum condition after IPTG induction.The proteins were expressed mainly as inclusion bodies with the yield of more than 30% of total bacterial proteins.The expressed products were purified through Ni~(2+)-affinity chromatographic column.After purificati...