Velkov Tony, Rimmer Kieran A, Headey Stephen J
School of Medicine, Deakin University, Pigdons Road, Geelong, 3217 Victoria, Australia.
Protein Expr Purif. 2010 Apr;70(2):260-9. doi: 10.1016/j.pep.2009.09.012. Epub 2009 Sep 24.
A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPAR alpha LBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPAR alpha LBD (aa196-468) was synthesized and ligated into the pDEST17 E. coli expression vector downstream of a MBP solubility fusion tag and an intermittent TEV protease cleavage site. Following auto-induction at 28 degrees C, PPAR alpha LBD protein was purified to electrophoretic homogeneity by a nickel affinity chromatographic step, on-column TEV protease cleavage followed by Sephacryl S200 size exclusion chromatography. The recombinant protein displayed cross-reactivity with goat anti-(human PPAR alpha) polyclonal antibody and was identified as human PPAR alpha by trypic peptide mass finger-printing. The addition of a PPAR alpha specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPAR alpha LBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPAR alpha LBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPAR alpha LBD construct make it amenable to high through-put screening assays in drug discovery programs.
一种人过氧化物酶体增殖物激活受体α配体结合域（PPARα LBD）-麦芽糖结合蛋白融合构建体在大肠杆菌中表达。合成了编码人PPARα LBD（第196至468位氨基酸）的密码子优化DNA序列，并将其连接到pDEST17大肠杆菌表达载体中，位于MBP溶解性融合标签和间歇性TEV蛋白酶切割位点的下游。在28℃进行自诱导后，通过镍亲和色谱步骤将PPARα LBD蛋白纯化至电泳纯，进行柱上TEV蛋白酶切割，然后进行Sephacryl S200尺寸排阻色谱。重组蛋白与山羊抗（人PPARα）多克隆抗体显示出交叉反应性，并通过胰蛋白酶肽质量指纹图谱鉴定为人PPARα。向生长培养基中添加PPARα特异性配体（非诺贝特酸、GW7647或GW590735）可显著稳定PPARα LBD结构并增强可溶性蛋白的表达。通过监测PPARα LBD表达随生长培养基中配体浓度的变化来检测细胞内配体结合。所报道的PPARα LBD构建体的高效表达和细胞内测定使其适用于药物发现计划中的高通量筛选测定。
