【摘要】 目的:以重组人角化细胞生长因子(recombinant human keratinocyte growth factor-2,rhKGF-2)为模型,研究一种对pH较为敏感的多聚化合物聚丙烯酸树脂EudragitS-100对蛋白折叠的促进作用及其作用机制。方法:直接将变性蛋白加入到含有不同浓度Eudragit的蛋白复性缓冲液中,采用MTT法、荧光分光光度法、圆二色光谱法以及高效液相色谱法来比较分析不同浓度EudragitS-100对变性rhKGF-2的复性促进作用。结果:在Eudragit S-100作用下,rhKGF-2的复性产率比普通稀释复性法显著增高且最高达到71%。研究表明Eudragit S-100的促进蛋白折叠的作用是基于Eudragit S-100与rh-KGF-2发生了特异性的结合反应。另外,本研究还发现蛋白复性缓冲液中尿素的浓度对蛋白的复性也存在较大影响。结论:Eudragit S-100能显著提高以rhKGF-2为代表的蛋白的正确折叠和复性收率。更多还原

【Abstract】 Aim:To study the effect of Eudragit S-100,a pH-responsive polymer,on protein refolding level,using recombinant human keratinocyte growth factor-2(rhKGF-2) as a model protein.Methods:The refolding of rhKGF-2 was performed by directly diluting denatured rhKGF-2 into a refolding buffer containing different concentrations of Eudragit.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTT assay,reverse phase HPLC,fluorescence emission spectroscopy and circular dichroism spectroscopy.Results:The addition of Eudragit S-100 in the refolding buffer significantly increased the rhKGF-2 refolding yield to 71%,when dilution refolding was conducted at 0.5 mg/mL rhKGF-2.The outcome from the refolding study showed possibility of a special interaction between rhKGF-2 and Eudragit,suggesting that the refolding-enhancing ability of Eudragit S-100 was due to this interaction between Eudragit S-100 and rhKGF-2.Mean while,the result showed that the concentration of urea was also an important factor for the optimization of the refolding in the presence of Eudragit.Conclusion:Eudragit S-100 can significantly increase the refolding level of rhKGF-2.更多还原