【作者】 陈亚坤； 陈枢青； 曾苏；

【Author】 CHEN Ya-Kun CHEN Shu-Qing ZENG Su~* (Dept.of Drug Metabolism & Pharmaceutical Analysis and Dept.of biopharmaceutics,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310006,China)

【机构】 浙江大学药学院药物分析教研室生物制药教研室；

【摘要】 从E.coli JM83基因组和人肝总RNA中分别扩增获得细菌碱性磷酸酶(BacterialAlkaliile Phosphatase,BAP)基因和人甾体受体辅活化因子-1(Steroid Receptor Coactivator-1,SRC-1)的186个氨基酸对应的基因序列(以下简称SRC186)。用重组技术,构建BAP-SRC186-pET28a 融合基因表达载体,在E.coli Rosetta(DE3)中,以IPTG 低温诱导表达,获得可溶性融合蛋白BAP-SRC186。表达实验结果表明,Rosetta(DE3)对该蛋白的表达能力强于BL21(DE3),BAP 的存在有利于可溶性SRC186的获得。以对硝基苯磷酸盐(p-nitrophenyl-phosphate,PNPP)为底物进行的活性测定显示,该融合蛋白的BAP 比活力为0.176±0.0134μmol/min/mg 蛋白。活性表达的BAP-SRC186融合蛋白被应用于辅活化因子与核受体的相互作用研究。在利福平存在的情况下,BAP-SRC186能与孕烷X 受体配体结合域(Pregnane X Receptor Ligand Binding Domain,PXRLBD)发生利福平剂量依赖性的相互作用,作用强度通过BAP 的显色反应可方便地检测。BAP 融合蛋白能有效应用于蛋白质相互作用研究。更多还原

【Abstract】 The bacterial alkaline phosphatase(BAP)gene and gene fragment corresponding to 186amines of human steroid receptor coactivator-1(SRC-1)were obtained respectively from E.coliJM83 and total RNA of human liver by PCR amplification.The correct fragments were subclonedinto pET28a to construct a BAP-SRC 186-pET28a expression plasmid.In E.coli Rosetta(DE3),the fusion protein BAP-SRC186 was expressed in soluble form after induction by IPTG in lowtemperature.Rosetta(DE3)was proved to be a more efficient expression host strain thanBL21(DE3)for this fusion protein,and BAP had helpful effects on soluble expression of SRC 186.The activity of BAP was 0.176±0.0134 μmol/min/mg protein measured by p-nitrophenyl-phosphate(PNPP)assay.Then the active fusion protein was applied into research of theinteraction between coactivator and nuclear receptor.In the presence of rifampin,BAP-SRC 186interacted with Pregnane X Receptor Ligand Binding Domain(PXRLBD)in rifampin dose-dependent way,and the protein-protein interactions were detected as activities of BAP.Itindicated the active BAP fusion protein could be used in interaction researches of protein-proteininteractions.更多还原