Examination of Three Alternative Devices to Refold Correctly the Denatured Enzyme Originated from the Precipitate Produced in the Process of Protein Synthesis

蛋白质复性是重组DNA蛋白质合成下游的关键过程之一。本研究的目的是比较评价以下三种方法使球状蛋白质从其随机卷曲结构中正确复性，例如在6MGdnHCl中，即(1)首先建立疏水核心，(2)通过在氨基末端固定化使其有望从其氨基末端折叠，(3)通过在羧基末端固定化使其有希望从氨基末端折叠。实验上，首先，疏水核心的形成是通过使用高离子强度的复性介质实现的；根据已知的方法，N或C末端(2)或(3)的固定化是通过共价键合在琼脂糖凝胶上的方式进行的。当然，这种不可逆的共价固定蛋白质可能不符合实际应用，然而，在蛋白质复性中，它是…更多地采用，以对上述三种装置进行更准确的评估。采用的球状蛋白是牛胰腺核糖核酸酶A(RNase)、蛋清溶菌酶(Lyzm)和细菌枯草杆菌毒素Bpn‘(SBTL)。在检测过程中发现RNase和Lyzm不适合本试验。由于分子内的酰胺化作用，核糖核酸酶不能被固定在保留活性形式的C末端，固定化的Lyzm对于以细胞壁为底物的通用活性测定程序是不能容忍的。幸运的是，通过使用Sbtl，有可能准确地检查上述三个装置的正确折叠。至于最初打算由疏水核心形成的装置(1)，我们成功地将6M GdnHCl变性的Sbtl在pH 2.4的条件下通过在1.5-2.0M的K-醋酸钾中在pH 6.5的条件下孵育而重新折叠。然而，蛋白水解酶Sbtl需要进行自我蛋白降解才能获得高达30%的最大复性产率。装置(2)或(3)分别对应于固定化的Sbtl的N端和C端的复性，其方法是将固定化的Sbtl在6M GdnHCl中反复溶解，然后在2M K-乙酸酯中进行复性。在这两种情况下，在第三次变性/复性循环后，复性产率几乎达到100%。在装置(2)的情况下，复性速率似乎大于装置(3)，而且，2M醋酸钾的复性介质优于2M KCl复性介质。总之，只要给出合适的复性环境，Sbt1在所有三种情况下都几乎实现了定量复性。其中，最重要的机制可能是在Sbt1的复性过程中形成疏水核心，然后是N端和C端的固定化。较少

Protein refolding is one of the critical processes in the downstream of the recombinant DNA protein synthesis. The objective of the present research work is to evaluate comparatively following three devices for the correct refolding of a globular protein from its random coil structure produced, for example, in 6M GdnHCl ; that is, (1) to build the hydrophobic core in the first place, (2) to let it refold hopefully from its carboxy-terminal by immobilization at the amino-terminal, (3) to let it refold hopefully from its amino-terminal by immobilization at the carboxy-terminal.Experimentally, hydrophobic core formation in the first place, (1), was realized by use of a refolding medium of high ionic strength ; immobilization of either N or C-terminal, (2) or (3), was conducted by means of covalently bonding on sepharose gel beads according to the known methods.Of course, such irreversible covalent fixation of the protein might not meet practical uses, in protein refolding, however, it was … More adopted to get more precise evaluation for the above-mentioned three devices. Adopted globular proteins were bovine pancreatic ribonuclease A(RNase), hen egg-white lysozyme(Lyzm), and bacterial subtilisin BPN'(Sbtl).It became clear in the course of examination that RNase and Lyzm were not suitable for the present test. RNase could not be immobilized at C-terminal retaining active form due to intramolecular amidation and immobilized Lyzm was not tolerable for the generalized activity assay procedure which uses cell walls as a substrate. Fortunately, by use of Sbtl, it was possible to examine exactly the above-mentioned three devices for the correct folding.As for the device (1) which intends to form by hydrophobic core at first, we succeeded to refold 6M GdnHCl-denatured Sbtl at pH 2.4 by incubating it in 1.5-2.0M K-acetate at pH 6.5. However, the protease Sbtl suffers from autoproteolytic digestion to get a resultant maximum refolding yield of up to 30%. A precise evaluation of this device was attained by deletion of an autolysis event through the immobilization as follows.Device (2) or (3) which corresponds to the refolding of Sbtl with N-terminal or C-terminal immobilized, respectively, was conducted by repeated dissolution of immobilized Sbtl in 6M GdnHCl followed by its refolding in 2M K-acetate. In both cases, almost 100% refolding yield was achieved after the 3rd denaturation/renaturation cycle based on the recovered activity of the preceding cycle.Rate of refolding in the case of device (2) appeared to be larger than that of device (3) Moreover, a refolding medium consisted of 2M K-acetate was superior to that of 2M KCl. In conclusion, almost quantitative refolding of Sbtl was achieved in all three cases, provided that a suitable refolding environment was given. Among them, the most important device might be a hydrophobic core formation in the refolding of Sbtl followed by N-terminal and C-terminal immobilization. Less