论文部分内容阅读
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。
To establish a urea-gradient full-length human PPAR-γ recombinant protein on a size-exclusion column. Dimensionally denaturing columns were used to directly denature the denatured proteins by using a urea gradient on a size-exclusion column. Addition of urea and arginine inhibits aggregation to increase renaturation yield, adding Zn2 + promotes denatured proteins to form correct folds. After renaturation, the size of the recombinant protein retained its size. The UV absorption maximum wavelength was changed from 234.0 nm to 280.8 nm. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry was used to analyze the integrity of the recombinant protein. The results showed that the dissociation constant (Kd) was 89 ± 4nmol / L and the yield of renaturation was 52.7%. The results showed that the method established in this paper can be successfully used to renature the full length human PPAR-γ, and to obtain the biologically active full-length human PPAR-γ protein which can be used for structural and functional studies.