尺寸排阻色谱柱上尿素梯度复性全长人PPAR-γ

建立尺寸排阻色谱柱上尿素梯度复性全长人PPAR-γ重组蛋白的方法。利用尺寸排阻色谱柱上尿素梯度除去变性剂直接复性变性蛋白。添加尿素和精氨酸抑制聚集以提高复性产率,加入Zn2+促进变性蛋白形成正确折叠。复性后,重组蛋白的尺寸排阻色谱保留体积增大;紫外光谱最大吸收波长从234.0nm变为280.8nm;基质辅助激光解析-电离飞行时间质谱分析重组蛋白的完整性,测得分子量与理论值一致;放射配体受体结合饱和实验测得解离常数(Kd)为89±4nmol/L;复性产率为52.7%。结果表明本文所建立的方法能成功用于复性变性全长人PPAR-γ,获得可用于结构和功能研究的具有生物学活性的全长人PPAR-γ蛋白。

On-Column Refolding of Full-Length Human PPAR-γ by Urea Gradient Size-Exclusion Chromatography

The on-column refolding of full-length human peroxisome proliferator-activated receptor-γ（PPAR-γ） was developed by urea gradient size-exclusion chromatography （SEC）.The unfolded protein was directly refolded on-column by urea gradient SEC for gradual removal of denaturants.The low molecular weight additives,such as urea and arginine were used to suppress aggregation of proteins for obtaining the good yield of refolding,and Zn2＋ was added to facilitate the formation of correct folding.After refolding,the retention volume of recombinant protein increased,and the maximum absorption wavelength of ultra violet spectroscopy（UV） changed from 234.0nm to 280.8nm.Matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI TOF-MS） analysis was performed to investigate the integrity of the refolded protein with the protonated molecule ion at an m/Z value in good agreement with the calculated molecular weight for the His6-PPAR-γ.The Kd value of 89±4nmol/L was determined by radio-ligand receptor binding assay and the yield of refolding was 52.7%.These results demonstrate that the developed strategy here can be used successfully to produce biologically active full-length human PPAR-γ for the investigation of structure and function.