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目的构建人第10号染色体上磷酸酶和张力蛋白同源缺失的基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)原核表达质粒,使其在原核细胞中高效表达并进行纯化。方法将全长片段插入原核表达载体pGEX-4T-1,构建重组子pGEX-4T-1-PTEN,转化BL-21感受态细胞。经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达PTEN融合蛋白的可诱导性表达,通过SDS-PAGE电泳、Western印迹分析证实蛋白表达的特异性。并用谷胱甘肽-S-转移酶(glutathione-Stransferase,GST)亲和层析对融合蛋白进行纯化。结果成功构建了原核表达载体pGEX-4T-1-PTEN,并将PTEN融合蛋白的成功表达,通过SDS-PAGE电泳、Western印迹分析,证实了蛋白表达的特异性。并对蛋白进行了纯化,获得了GST-PTEN融合蛋白的纯品。结论成功表达、纯化了GST-PTEN融合蛋白,为进一步研究PTEN蛋白的功能及其与其它功能性蛋白的相互作用研究奠定了基础。
Objective To construct a prokaryotic expression plasmid of phosphatase and tensin homology deleted on chromosome ten (PTEN) on human chromosome 10 and to express it in prokaryotic cells for purification. Methods The full-length fragment was inserted into prokaryotic expression vector pGEX-4T-1 to construct recombinant pGEX-4T-1-PTEN and transformed into BL-21 competent cells. Inducible expression of the PTEN fusion protein was induced by isopropylthio-β-D-galactoside (IPTG), and the specificity of the protein expression was confirmed by SDS-PAGE electrophoresis and Western blot analysis. The fusion protein was purified by glutathione-Stransferase (GST) affinity chromatography. Results The prokaryotic expression vector pGEX-4T-1-PTEN was successfully constructed and the PTEN fusion protein was successfully expressed. The specificity of the protein expression was confirmed by SDS-PAGE electrophoresis and Western blot analysis. The protein was purified and the pure GST-PTEN fusion protein was obtained. Conclusion The GST-PTEN fusion protein was successfully expressed and purified, which laid the foundation for the further study on the function of PTEN protein and its interaction with other functional proteins.