| 蛋白酪氨酸磷酸酶PTP1B催化活性区的原核表达及活性分析 |
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| 引用本文： | 王生育,颜江华,潘阳霖,李学军,陈忠. 蛋白酪氨酸磷酸酶PTP1B催化活性区的原核表达及活性分析[J]. 微生物学报, 2008, 24(4): 553-557 |
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| 作者姓名： | 王生育 颜江华 潘阳霖 李学军 陈忠 |
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| 作者单位： | 厦门大学医学院抗癌研究中心, 厦门 361005;厦门大学医学院抗癌研究中心, 厦门 361005;厦门大学生物医学工程研究中心, 厦门 361005;厦门第一医院, 厦门 361001;厦门大学生物医学工程研究中心, 厦门 361005 |
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| 基金项目： | 厦门市重大疾病攻关研究基金(No. 3502Z20051027)和卫生部(福建省)卫生教育联合攻关项目(No. Wkj2005-2-019)资助。 |
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| 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。
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| 关 键 词： | PTP1Bc 基因表达 蛋白纯化 活性分析 |
Expression and Activity Analysis of Catalytic Domain of PTP1B |
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| Shengyu Wang,Jianghua Yan,Yanglin Pan,Xuejun Li and Zhong Chen. Expression and Activity Analysis of Catalytic Domain of PTP1B[J]. Acta microbiologica Sinica, 2008, 24(4): 553-557 |
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| Authors: | Shengyu Wang Jianghua Yan Yanglin Pan Xuejun Li Zhong Chen |
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| Affiliation: | Cancer Research Center of Medical College, Xiamen University, Xiamen 361005, China;Cancer Research Center of Medical College, Xiamen University, Xiamen 361005, China;Research Center of Biomedical Engineering, Xiamen University, Xiamen 361005, China;The First Hospital of Xiamen, Xiamen 361001, China;Research Center of Biomedical Engineering, Xiamen University, Xiamen 361005, China |
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| 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. |
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| Keywords: | PTP1Bc expression protein purification activity analysis |
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