固相金属亲和层析纯化全长人PPARγ重组蛋白

Purification of Full Length Human PPARγRecombinant Protein by Immobilized Metal-ion Affinity Chromatography

建立全长人PPARγ重组蛋白的固相金属亲和层析纯化方法。利用E.coli BL_(21)(DE3)细胞外源性表达出全长人PPARγ重组蛋白,采用固相金属亲和层析法纯化目标蛋白。通过考察咪唑浓度对纯化的影响,确定在8 mol/L尿素下,用含50 mmol/L咪唑的缓冲液冲洗,200mmol/L咪唑缓冲液洗脱,可获得纯度为95%的目标蛋白,透析复性后经放射配体受体饱和结合实验测定全长人PPARγ重组蛋白的解离常数K_d为106±6nmol/L。结果表明本文所建立的方法能够成功纯化出高纯度的目标蛋白,经复性后,可获得用于结构和功能研究的全长人PPARγ重组蛋白。

A strategy for purification full length human peroxisome proliferator activated receptor-γ（PPARγ）was developed by immobilized metal-ion affinity chromatography. The full length human PPAR7 Recombinant Protein was expressed by E. coli BL21 （DE3） ;and purification was carried out by immobilized metal-ion affinity chromatography. The conditions： 50mmol/L imidazole in washing buffer and 200mmol/L imidazole in elution buffer with 8mol/L Urea,that was optimized by examining imidazole concentration. 95% purity of the full length PPARγ was obtained by one-step purification with IMAC. After dialysis refolded ,the activity was tested with radio-ligand receptor binding assay and the Kd was 106 ± 6nmol/L. These results demonstrate that the strategy here can be used to produce biologically activie full length human PPARγ recombinant protein for the investigation of structure and function.