Application of Immobilization Method to Mechanistic Study and Continuous Procedure of Protein Refolding

固定化方法在蛋白质重折叠机理研究和连续过程中的应用

• 批准号: 08650951
• 负责人: NOHARA Daisuke
• 金额: $ 1.41万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08650951/
• 关键词: Protein; Refolding; Immobilization

(1) In order to select protein refolding media, two different types of immobilized subtilisin BPN' were prepared. One was immobilized on agarose beads by the amino groups of the enzyme and the other was immobilized on agarose beads by the carboxyl groups. Using the quantitatively renaturing preparations obtained by repeated denaturation/renaturation treatment of the immobilized subtilisin, it was revealed that both of two immobilized preparations were useful for media selection.(2) By use of the quantitatively renaturing preparations of subtilisin and thermolysin, organic acids such as 2 M potassium acetate and potassium propionate were found to be excellent media. These solvents were not at all effective to the refolding of hen egg-white Lysozyme.(3) In the case of Streptomyces griseus trypsin (SGT) that involves 3 disulfide bonds in a molecule, the quantitatively renaturing preparation could not be obtained with agarose beads probably because the denaturation/renaturation treatment involved the redox reaction. Instead, immobilization of SGT on glass beads brought about the satisfactory result.(4) In the presence of immobilized benzamidine that represents inhibitory effect to SGT, the refolding yield of SGT in solution system was considerably increased.(5) This fact made it possible to design a continuous and a large-scale refolding of SGT by use of the immobilized inhibitor.

(1)为了筛选蛋白质复性介质，制备了两种不同类型的固定化枯草杆菌蛋白酶BPN'。一种是通过酶的氨基固定在琼脂糖珠上，另一种是通过羧基固定在琼脂糖珠上。利用固定化枯草杆菌蛋白酶反复变性/复性处理得到的定量复性制剂，发现两种固定化制剂都可用于培养基的选择。(2)利用枯草杆菌蛋白酶和嗜热菌蛋白酶的定量复性制剂，发现有机酸如2 M乙酸钾和丙酸钾是优良的介质。这些溶剂对溶菌酶的复性效果不明显。(3)在灰色链霉菌胰蛋白酶（SGT）的情况下，涉及3个二硫键在一个分子中，定量复性制备不能得到与琼脂糖珠可能是因为变性/复性处理涉及氧化还原反应。相反，SGT固定在玻璃珠上带来了令人满意的结果。(4)固定化苯甲脒对SGT有抑制作用，在此作用下，SGT在溶液体系中的复性率显著提高。(5)这一事实使得有可能设计一个连续的和大规模的SGT的复性通过使用固定化的抑制剂。

项目成果

Daisuke Nohara: "High Performance in Refolding of Streptomyces griseus Trypsin by the Aid of a Mutant of Streptomyces Subtilisin Inhibitor……" Journal of Biochemistry. 125. 343-347 (1999)

Daisuke Nohara：“借助链霉菌枯草杆菌蛋白酶抑制剂突变体实现灰色链霉菌胰蛋白酶的高效重折叠……”《生物化学杂志》125. 343-347 (1999)。

Daisuke Nohara: "Fundamental Conditionings for Refolding of Denatured Proteins" Recent Research Developments in Chemical and Pharmaceutical Sciences. 1. 79-89 (1996)

Daisuke Nohara：“变性蛋白质重折叠的基本条件”化学和制药科学的最新研究进展。

Daisuke Nohara: "A New Method of Media Selection for Protease Refolding by Application of Immobilized Subtilisin Preparation" Journal of Fermentation and Bioengineering. 82. 315-318 (1996)

Daisuke Nohara：“应用固定化枯草杆菌蛋白酶制剂进行蛋白酶重折叠的培养基选择新方法”发酵与生物工程杂志。

Daisuke Nohara: "Kinetic Study on Thermal Denaturation of Hen Egg-White Lysozyme Involving precipitation" Journal of Bioscience and Bioengineering. 87. 199-205 (1999)

Daisuke Nohara：“涉及沉淀的鸡蛋清溶菌酶热变性的动力学研究”生物科学与生物工程杂志。

Daisuke Nohara: "Design of optimum refolding solution by combination of reagents classified by specific function" Journal of Fermentation and Bioengineering. 82・4. 401-403 (1996)

野原大辅：“通过按特定功能分类的试剂组合设计最佳重折叠溶液”《发酵与生物工程杂志》82・4（1996）。