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

近年来，我们基于分子伴侣的功能，开发了一种利用缓慢透析使变性和还原蛋白恢复的有效方法。该方法可有效地将蛋清溶菌酶和单克隆抗体（IgG1/kappa）从其变性和还原形态中还原出来。本课题的目的是研究复复性方法是否适用于其他变性和还原蛋白的复复性。首先，由于一种变性和还原的单克隆抗体（IgG1/kappa）可以通过缓慢透析的方法有效地恢复到完整状态，我在体外分析了变性和还原形式的单克隆抗体的恢复机制。结果表明，折叠后的重链与轻链相互作用，形成完整形态。其次，缓慢透析方法在变性和还原溶菌酶的有效复性方面具有优势，这些溶菌酶是从大肠杆菌中表达的，或者蛋氨酸残基被标记为^<13>细胞核。此外，该方法适用于变性和还原的单体蛋白如核糖核酸酶A、take -淀粉酶A和烯醇化酶等低聚蛋白的有效复性。对于不稳定的蛋白质，在还原缓冲液中加入甘油和硫酸铵也被证明是有效的。

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.