摘要
目的获得活性表达的细菌碱性磷酸酶(bacterial alkaline phosphatase,BAP)基因和人甾体受体辅活化因子-1(steroid recep- tor coactivator-1,SRC-1)的融合蛋白,应用于辅活化因子与核受体的结合研究。方法从Escherichia coli JM83基因组和人肝总RNA中分别扩增获得BAP基因和SRC-1的186个氨基酸对应的基因序列(简称SRC186)。用重组技术,构建BAP-SRC186- pET28a融合基因表达载体,在Escherichia coli Rosetta(DE3)中,以IPTG低温诱导表达。以对硝基苯磷酸盐(p-nitrophenyl-phos- phate,PNPP)为底物进行活性测定。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结合研究。结果获得可溶性融合蛋白BAP-SRC186。该融合蛋白的BAP比活为(0.176±0.013 4)μmol·min-1·mg(pro)-1 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结论BAP融合蛋白与核受体的结合研究为药物代谢酶调控的体外研究开辟了新的思路。
OBJECTIVE To obtain the active fusion protein in of bacterial alkaline phosphatase（BAP） and human steroid receptor coaetivator-1 （ SRC-1 ） and apply this fusion protein in the regulation of drug metabolism enzymes. METHODS BAP gene and gene fragment corresponding to 186 amines of SRC-1 were obtained respectively from the genome of Escherichia coli JM83 and total RNA of human liver by PCR amplification. The correct fragments were subcloned into pET28a to construct an expression plasmid of BAP-SRC186-pET28a. In Escherichia coil Rosetta（DE3）, the fusion protein of BAP-SRCI86 was expressed after being induced by IPTG at low temperature. The interaction between coactivator and nuclear receptor was studied with the active fusion protein. RESULTS The activity of BAP was （0.176 ± 0.013 4）μmol·min^-1·mg（pro）^-1 measured by p-nitrophenyl-phosphate（PNPP） assay. In the presence of rifampicin, BAP-SRC186 interacted with pregnane X receptor ligand binding domain（PXRLBD） in rifampiein dose-dependent pattern, and the protein-prutein interactions were detected by the activity of BAP. CONCLUSION The active fusion protein BAP-SRC-1 could be used in the regulation research of drug metabolism enzymes.
出处
《中国药学杂志》
EI
CAS
CSCD
北大核心
2005年第15期1192-1195,共4页
Chinese Pharmaceutical Journal
基金
国家自然科学基金项目(30472171
30225047)
