共表达PXRLBD和SRC88以及PXR配体筛选的平衡透析模型的建立

孕烷X受体(pregnane X receptor,PXR),属核受体NR1I亚家族.PXR可由配体活化,当配体(如外源药物)与其配体结合域(ligand binding domain,LBD)结合后,PXR被活化,招募辅调控因子(如人甾体受体辅活化因子-1,steroid receptor eoactivator-1,SRC-1)形成复合物,通过其DNA结合域(DNA binding domain)结合到药物代谢酶基因启动子的特定DNA序列上,从而调控CYP3A4等药物代谢酶基因的转录1,2].

Coexpression of PXRLBD with SRC88 and construction of equilibrium dialysis model of screening PXR ligands

The aim of this study was to obtain the soluble protein of human pregnane X receptor ligand binding domain (PXRLBD) through the coexpression of PXRLBD and 88 amino acids of steroid receptor coactivator-1 (SRC88) and apply the protein to constructing a new model of screening PXR ligands. Expression plasmid of pETDuet-1-SRC88-PXRLBD was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88 via induction by IPTG at low temperature. Then an equilibrium dialysis model was constructed to study the interaction between PXRLBD and drugs including clotrimazole and dexamethasone, using HPLC as the analysis method. The results showed that the soluble protein of PXRLBD was obtained and the HPLC data indicated that clotrimazole bound to PXRLBD, while dexamethasone did not bind to PXRLBD, which indicated the successful establishment of a new method for studying the interaction between PXR and drugs. The new method may be useful in the screening of PXR ligands in vitro.