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

基于生物学功能的变性蛋白有效复性方法的构建

• 批准号: 08558072
• 负责人: UEDA Tadashi
• 金额: $ 5.31万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08558072/
• 关键词: protein; folding; dialysis; immunoglobulin G; lysozyme; リボヌクレアーゼA; タカアミラーゼA; エノラーゼ; 単鎖Fv; インクルージョンボディ; クエン酸シンターゼ

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.

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。

项目成果

Yoshitake Maeda: "Effect of additives on the renaturation of reduced lysozyme in the presence of 4M urea." Protein Eng.9. 461-465 (1996)

Yoshitake Maeda：“在 4M 尿素存在下，添加剂对还原溶菌酶复性的影响。”

Y.Abe et al.: "An improved method for preparing lysozyme with chemically ^<13>C-enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis" J.Biochem.122. 1153-1159 (1997)

Y.Abe等人：“使用2-氨基苯硫酚作为硫解试剂制备具有化学上13 C富集的甲硫氨酸残基的溶菌酶的改进方法”J.Biochem.122。

T.Ueda et al.: "Identification of the peptide region that folds native conformation in the early stage of the renaturation of reduced lysozyme." Biophys. Biochem.Res.Commun.228. 203-208 (1996)

T.Ueda 等人：“鉴定在还原型溶菌酶复性早期折叠天然构象的肽区域。”

S.Mine et al.: "Improvement of the refolding yield and solubility of hen egg-while lysozyme by altering Met residue attached to its N-terminal to Ser." Protein Eng. 10. 1333-1338 (1997)

S.Mine 等人：“通过将 N 端连接的 Met 残基改为 Ser，提高鸡蛋溶菌酶的重折叠产量和溶解度。”

Tadashi Ueda: "Favourable interaction between heavy and light chains arrests the undesirable ollgomerization of heavy chains in the refolding of denatured and reduced immunogloblinG" Cell.Mol.Life.Sci.53. 929-934 (1997)

Tadashi Ueda：“重链和轻链之间的有利相互作用阻止了变性和还原免疫球蛋白重折叠过程中不希望出现的重链低聚化”Cell.Mol.Life.Sci.53。