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

(1)本研究的目的是设计重组DNA法产生的沉淀蛋白正确折叠到其真实3d结构的工艺。另一个目的是检查诱导再折叠的可能性，即测试是否有任何物质可以增强再折叠。(2)本研究采用完整的蛋清溶菌酶（Lyzm）和完全还原的Lyzm制剂。通过CD测定和酶活性测定来评价再折叠率。(3)具有4个S-S键的完整Lyzm溶解在变性剂溶液中，如6M氯化胍（Gdn-HC1）中，通过CD测量证实其处于随机线圈状态。CD也显示100%的未折叠Lyzm通过稀释到浓度。恢复的酶活性证实了同样的结果。6M尿素+ 6M LiCl溶液对Lyzm的变性效果与6M Gdn-HCl相同。这意味着一个双功能变性剂被分为两个单功能变性剂。因此，尿素和LiCl可以独立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Lyzm的再折叠过程中，建立了四个对再折叠非常有用的基本程序和/或条件：1)低浓度的蛋白质；2)维持蛋白质在松散折叠状态下的溶液，例如在2M尿素中；3)建议延迟氧化，或在松散折叠后形成S-S键；4)在整个过程中降低温度，以夸大正确结构和错误结构之间的小能量差。结果，根据成熟溶液中[Lyzm] = 1.1uM时的恢复活性，我们获得了95%以上的再折叠收率。遗憾的是，到目前为止，我们还不知道可能的物质可以增强Lyzm的再折叠。我们正试图找到能诱导Lyzm以外的酶再折叠的有效物质。我们相信，在这项研究工作中建立的一些基础知识将有助于蛋白质重折叠这一令人着迷的研究领域的发展。少

(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