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

三种替代装置正确重折叠蛋白质合成过程中产生的沉淀物变性酶的检验

基本信息

批准号：
03555180
负责人：
SAKAI Tomoya
金额：
$ 1.6万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

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

蛋白质重折叠是重组DNA蛋白质合成下游的关键过程之一。目前研究工作的目的是比较评估以下三种设备，以从其产生的随机卷曲结构中正确地重折叠球状蛋白，例如在 6M GdnHCl 中；也就是说，（1）首先构建疏水核心，（2）通过固定在氨基末端，使其有希望从其羧基末端重折叠，（3）通过固定在羧基末端，使其有希望从其氨基末端重折叠。在实验上，首先，疏水核心的形成，（1）是通过使用高离子重折叠介质实现的力量 ; N端或C端（2）或（3）的固定是根据已知的方法通过共价键合在琼脂糖凝胶珠上进行的。当然，蛋白质的这种不可逆共价固定可能不满足实际用途，在蛋白质重折叠中，然而，为了对上述三种装置进行更精确的评估，采用了这种方法。采用的球状蛋白为牛胰核糖核酸酶A（RNase）、鸡蛋清溶菌酶（Lyzm）和细菌枯草杆菌蛋白酶BPN'（Sbtl）。在检查过程中发现RNase和Lyzm不适合本试验。由于分子内酰胺化，RNase 无法固定在 C 端保留活性形式，并且固定化的 Lyzm 不能用于使用细胞壁作为底物的通用活性测定程序。幸运的是，通过使用Sbtl，可以准确地检查上述三种装置的正确折叠情况。对于最初打算由疏水核形成的装置(1)，我们通过将其在pH 6.5的1.5-2.0M K-乙酸盐中孵育，成功地在pH 2.4下重新折叠6M GdnHCl变性的Sbtl。然而，蛋白酶 Sbtl 会遭受自蛋白水解消化，从而获得高达 30% 的最大重折叠率。该装置的精确评估是通过如下固定化删除自溶事件来实现的。装置(2)或(3)分别对应于N端或C端固定化的Sbtl的重折叠，通过将固定化的Sbtl重复溶解在6M GdnHCl中，然后将其在2M K-乙酸盐中重折叠来进行。在这两种情况下，基于前一循环恢复的活性，在第三个变性/复性循环后实现了几乎 100% 的重折叠率。装置 (2) 的重折叠速率似乎大于装置 (3)。此外，由 2M K-乙酸盐组成的重折叠介质优于 2M KCl 的重折叠介质。总之，只要给定合适的重折叠环境，所有三种情况下都能实现几乎定量的 Sbtl 重折叠。其中，最重要的装置可能是 Sbtl 重折叠过程中疏水核心的形成，然后是 N 端和 C 端固定。较少的