目的研究小分子脲对包涵体蛋白质体外复性与同时纯化的作用。方法考察在高效疏水相互作用色谱(HPHIC)流动相中添加脲时,不同浓度的小分子脲对包涵体蛋白重组人酪氨酸激酶配体(recombined human Flt3ligand,rhFL)复性效率的影响,并采用...目的研究小分子脲对包涵体蛋白质体外复性与同时纯化的作用。方法考察在高效疏水相互作用色谱(HPHIC)流动相中添加脲时,不同浓度的小分子脲对包涵体蛋白重组人酪氨酸激酶配体(recombined human Flt3ligand,rhFL)复性效率的影响,并采用荧光光谱技术,进一步研究目标蛋白Flt3复性前后复性效果变化与荧光强度变化的规律。结果当脲的添加浓度为3mol·L^(-1)时,rhFL的质量回收率达到33.1%,更易于恢复其天然构象及生物活性。结论添加适当浓度的脲可促进rhFL的复性与同时纯化,通过荧光光谱技术可以初步推测包涵体蛋白在复性过程中生物活性的变化,为rhFL蛋白药物的研发提供参考。展开更多
Objective To characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes.To establish a spectral method which examine the extent of...Objective To characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes.To establish a spectral method which examine the extent of recombinant allergen renaturation.Methods The refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism,fluorescence and synchronous fluorescence spectra.IgE-binding capacity of recombinant protein was analyzed by ELISA.In addition,bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.Results Renaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.Conclusion The degree of protein renaturation correlated with the IgE-binding capacity of the protein.Results from this study may be of help for food allergy therapy and development of vaccination in the future.展开更多
文摘目的研究小分子脲对包涵体蛋白质体外复性与同时纯化的作用。方法考察在高效疏水相互作用色谱(HPHIC)流动相中添加脲时,不同浓度的小分子脲对包涵体蛋白重组人酪氨酸激酶配体(recombined human Flt3ligand,rhFL)复性效率的影响,并采用荧光光谱技术,进一步研究目标蛋白Flt3复性前后复性效果变化与荧光强度变化的规律。结果当脲的添加浓度为3mol·L^(-1)时,rhFL的质量回收率达到33.1%,更易于恢复其天然构象及生物活性。结论添加适当浓度的脲可促进rhFL的复性与同时纯化,通过荧光光谱技术可以初步推测包涵体蛋白在复性过程中生物活性的变化,为rhFL蛋白药物的研发提供参考。
基金supported by the Natural Science Foundation of China (30871752)the High-tech Industrialization Funding of Guangdong Province (2009B011300010)
文摘Objective To characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes.To establish a spectral method which examine the extent of recombinant allergen renaturation.Methods The refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism,fluorescence and synchronous fluorescence spectra.IgE-binding capacity of recombinant protein was analyzed by ELISA.In addition,bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.Results Renaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.Conclusion The degree of protein renaturation correlated with the IgE-binding capacity of the protein.Results from this study may be of help for food allergy therapy and development of vaccination in the future.
