Correct Refolding of Precipitated Enzymes Obtained in Recombinant DNA Process by Means of Induction Devices

重组 DNA 过程中通过感应装置获得的沉淀酶的正确重折叠

• 批准号: 02670983
• 负责人: SAKAI Tomoya
• 金额: $ 0.38万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670983/
• 关键词: Globular Protein; Lysozyme; Protein Folding; Protein Structure; Protein Aggregation; Protein Fixation; タンパク質三次元構造; タンパク質の変性; リフォ-ルディング; アンフォ-ルディング; アグリゲ-ション

(1) Objective of the present study is to design the process for refolding precipitated protein produced by the recombinant DNA method correctly to its authentic 3D-structure. Another aim is to examine possibility of induced refolding, i.e., to test if there is any substance that enhance refolding.(2) Intact hen egg-white lysozyme(Lyzm) and the fully reduced Lyzm preparation were adopted in the present research. Refolding yield was evaluated by CD measurement as well as by the recovered enzyme activity.(3) Intact Lyzm with four S-S bonds was confirmed to be in the random coil state by CD measurement when dissolved in the denaturant solution, e.g., 6M guanidinium chloride(Gdn-HC1). CD also showed 100% refolding of the unfolded Lyzm by mere dilution to concentrations. Recovered enzyme activity confirmed the same result.The 6M urea + 6M LiCl solution was as effective as 6M Gdn-HCl for denaturation of Lyzm. This implies that one bifunctional denaturant was divided into two monofunctional de … More naturants. Thus urea and LiCl can be used independently to contribute the diversity of denaturant selection encountered in the protein refolding process.In the course of refolding of the fully reduced Lyzm, four basic procedures and/or conditions were established as quite useful for the refolding : 1) low concentration of protein, 2) sustain the solution at loose folding state of protein, e.g. in 2M urea, 3) delayed oxidation, or S-S bond formation after the loose folding, is recommended, 4)lower temperature throughout the process to exaggerate small energy difference between the correct structure and incorrect structure. As a result we achieved more than 95% refolding yield based on recovered activity at [Lyzm] = 1.1uM in the maturing solution.Regretfully, we have no idea by now about possible substances that enhance refolding of Lyzm. We are trying to meet the effective substances that induce refolding of the other enzymes than Lyzm. We believe that several basic knowledges established in this research work will contribute to the development of the fascinating research field of protein refolding. Less

(1)本研究的目的是设计重组DNA方法产生的沉淀蛋白正确折叠到其真实的三维结构的过程。另一个目的是检查诱导重折叠的可能性，即，来测试是否有任何物质能增强重折叠。(2)本研究采用完整的鸡蛋清溶菌酶（Lyzm）和完全还原的Lyzm制剂。通过CD测量以及通过回收的酶活性来评价复性产率。(3)当溶解在变性剂溶液中时，通过CD测量证实具有四个S-S键的完整Lyzm处于无规卷曲状态，例如，6 M氯化胍（Gdn-HCl）。CD也显示了100%的复性展开的Lyzm仅稀释到浓度。用6 M尿素+6M氯化锂溶液对Lyzm进行变性，其效果与6 M盐酸胍溶液相同。这意味着一个双功能变性剂被分成两个单功能变性剂， ...更多信息 自然人因此，尿素和LiCl可以独立地用于蛋白质重折叠过程中遇到的变性剂选择的多样性。在完全还原的Lyzm的重折叠过程中，建立了四个基本程序和/或条件，对于重折叠非常有用：1）低浓度的蛋白质，2）将溶液维持在蛋白质的松散折叠状态，例如在2 M尿素中，3）延迟氧化，或松散折叠后的S-S键形成，4）在整个过程中降低温度，以夸大正确结构和不正确结构之间的微小能量差。结果，我们在成熟溶液中[Lyzm] = 1.1 μ M时获得了超过95%的复性率。遗憾的是，我们现在还不知道可能的物质可以增强Lyzm的复性。我们正试图满足诱导Lyzm以外的其他酶的重折叠的有效物质。我们相信，在这项研究工作中建立的几个基础知识将有助于蛋白质复性这一迷人的研究领域的发展。少