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将重组DNA法生产的沉淀蛋白正确复性为其真实三维结构的工艺。另一个目的是检测诱导复性的可能性，即测试是否有促进复性的物质。(2)本研究采用完整的蛋清溶菌酶(Lyzm)和完全还原的Lyzm制剂。(3)CD测定结果表明，含有4个S-S键的完整Lyzm在6M氯化胍(GdN-HCl)变性剂溶液中呈无规卷曲状态。CD还显示，仅稀释到浓度即可100%复性未折叠的Lyzm。6M尿素+6M氯化锂溶液对Lyzm的变性效果与6M GDN-HCl相当。这意味着一个双官能团变性剂被分成两个单官能团De…更多的自然人。在完全还原的LYZM的复性过程中，建立了四个对复性非常有用的基本程序和/或条件：1)低浓度的蛋白质，2)使溶液保持在蛋白质的松散折叠状态，例如在2M尿素中，3)延迟氧化，或松散折叠后S-S键的形成，4)整个过程中较低的温度，以夸大正确结构和错误结构之间的微小能量差异。结果，在成熟的溶液中，基于[Lyzm]=1.1微米的恢复活性，我们获得了95%以上的复性产率。遗憾的是，到目前为止，我们还不知道可能的物质促进了Lyzm的复性。我们正在努力寻找诱导Lyzm以外的其他酶重新折叠的有效物质。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折叠这一引人入胜的研究领域的发展。较少

(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