Construction of an effective renaturation method of denatured proteins based on a biological function

最近，我们基于分子伴侣的功能，开发了一种利用慢透析技术对变性和还原蛋白质进行有效复性的方法。该方法适用于蛋清溶菌酶和单抗(IgG1/kappa)的变性和还原复性。本项目的目的是研究复性方法是否适用于变性和还原的其他蛋白质的复性。首先，利用缓慢透析法将变性还原的单抗(IgG1/kappa)有效复性为完整的单抗，分析了变性还原单抗的体外复性机理。结果发现，折叠的重链和轻链之间的良好相互作用形成了完整的形式。其次，缓慢透析法在变性和还原的溶菌酶的复性方面具有优势，这些变性和还原的溶菌酶已在大肠杆菌中表达或蛋氨酸残基已被^&lt；13&gt；C标记。此外，该方法还适用于核糖核酸酶A、淀粉酶A等变性还原单体蛋白和烯醇化酶等寡聚蛋白的有效复性。对于不稳定蛋白质的复性，在复性缓冲液中加入甘油和硫酸铵也是有效的。

Recently, we have developed the effective renaturation method of denatured and reduced proteins using slow dialysis based on the function of a molecular chaperone. The method was applicable to renature both hen egg-white lysozyme and monoclonal antibody (IgG1/kappa) effectively from their denatured and reduced form. The aim of this project is to investigate whether or not the renaturation method is applicable to renature the denatured and reduced the other proteins. First, since a denatured and reduced monoclonal antibody (IgG1/kappa) effectively renatured to intact by use of the slow dialysis method, I analyzed a renaturation mechanism of the monoclonal antibody from denatured and reduced form in vitro. As a result, intact form was found to form by the favorable interaction between folded heavy chain and light chain. Next, the slow dialysis method was shown to have an advantage in the effective renaturation of denatured and reduced lysozyme, which had been expressed from E.coli or in which methionine residues had been labeled with ^<13>Cnuclei. Moreover, the method was applicable to the effective renaturation of denatured and reduced monomeric protein such as ribonuclease A and Take-amylase A and oligomeric protein such as enolase. For the renaturation of unstable proteins, the addition of glycerol and ammoniumsulfate to the ranturation buffer was also shown to be effective.