蛋白酪氨酸磷酸酶PTP1B催化活性区的原核表达及活性分析

Expression and Activity Analysis of Catalytic Domain of PTP1B

摘要:

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。

Abstract:

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 Ni2+-affinity chromatographic column. After purification, the purity of the proteins was more than 95%. Western blotting analysis suggested that the purified proteins could specially combine with anti-PTP1B antibody. Enzyme activity assay showed that the protein has phosphatase activities.