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

最近，我们开发了基于分子伴侣功能的变性和还原蛋白质的有效复性方法，使用缓慢透析。该方法适用于变性和还原的溶菌酶和单克隆抗体（IgG 1/kappa）的复性。本课题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，由于变性还原的单克隆抗体（IgG 1/κ）通过使用缓慢透析法有效地复性为完整，所以我分析了体外变性还原形式的单克隆抗体的复性机制。结果，发现通过折叠重链和轻链之间的有利相互作用形成完整形式。其次，缓慢透析法显示出在变性和还原溶菌酶的有效复性方面具有优势，所述变性和还原溶菌酶已从大肠杆菌表达或其中甲硫氨酸残基已用^<13>C核标记。此外，该方法适用于变性和还原的单体蛋白质如核糖核酸酶A和Take-amylase A以及寡聚蛋白质如烯醇化酶的有效复性。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.