Application of Molecular Chaperone to Protein Engineering

分子伴侣在蛋白质工程中的应用

基本信息

批准号：
06558097
负责人：
ESAKI Nobuyoshi
金额：
$ 3.78万
依托单位：
KYOTO 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：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06558097/
关键词：
inclusion body glutamate racemase alanine racemase molecular chaperone GroESL protein engineering

项目摘要

Overproduced recombinant proteins are often obtained in the form of inclusion bodies in the host cell, and it is sometimes difficult to solubilize the inclusion bodies into active forms in vitro. Recently, It has been reported that coexpression of the genes of molecular chaperones, dnaK and groESL,caused an increase in solubility of human procollagenase produced in E.coli. The aim of this study is to establish the method to prevent the recombinant protein from the inclusion body formation by the co-production of molecular chaperones. The overexpression of the murI (glr) gene, which encodes the glutamate racemase of Escherichia coli, resulted in the formation of inclusion bodies of the enzyme, and little activity was found in the soluble fraction of the transformant cells. The coexpression of the groESL gene with murI caused an in vivo solubilization of glutamate racemase in an active form. The solivility of glutamate racemase depends on the time and amount of the expression of GroESL g … More ene. In this study, we also examined the effect of GroESL on the folding of the domain peptide of bacterial alanine racemase. A subunit of thermostable alanine racemase of Bacillus stearothermophilus, a homodimer protein, is composed of two domains. When the genes encoding the N- and C-terminal peptide fragments corresponding to the domains were either in tandem or separately expressed in the same host cells, the active fragmentary enzyme was produced. However, when either the N or C-terminal fragment gene was alone expressed, the N-terminal fragment containing lysine 39 bound with the cofactor pyridoxal 5'-phosphate was mostly produced only in an insoluble form, and little C-terminal one was found. The soluble N-terminal fragment was produced on co-production with a molecular chaperone, GroESL,and showed alanine racemase activity. The denatured N-terminal fragment restored the activity on refolding with GroESL.Thus, only the N-terminal domain is involved in catalysis, and the C-terminal one functions as a kind of intramolecular chaperone to help the N-terminal one to fold correctly and can be functionally replaced by GroESL. Less

过量生产的重组蛋白在宿主细胞中往往以包涵体的形式存在，有时很难在体外将包涵体溶解成活性形式，最近有报道称分子伴侣、dnaK和groESL基因共表达。，导致大肠杆菌中产生的人原胶原酶的溶解度增加。本研究的目的是建立防止重组蛋白形成包涵体的方法。编码大肠杆菌谷氨酸消旋酶的 murI (glr) 基因的过度表达导致了该酶的包涵体的形成，并且在转化体的可溶部分中发现了很少的活性。 groESL 基因与 murI 的共表达导致活性形式的谷氨酸消旋酶的体内溶解。消旋酶取决于 GroESL g … More ene 的表达时间和量。在这项研究中，我们还研究了 GroESL 对嗜热脂肪芽孢杆菌热稳定性丙氨酸消旋酶亚基折叠的影响。同二聚体蛋白由两个结构域组成，当基因编码对应于结构域的N端和C端肽片段时，这些结构域串联或单独表达。然而，当N端或C端片段基因单独表达时，含有与辅因子吡哆醛5'-磷酸结合的赖氨酸的N端片段大多仅在相同的宿主细胞中产生。发现了一种不溶性形式，并且发现了少量的可溶性 N 端片段，该片段是与分子伴侣 GroESL 共同产生的，并显示出丙氨酸消旋酶活性。变性的N端片段恢复了GroESL重折叠的活性。因此，只有N端结构域参与催化，而C端结构域作为一种分子内伴侣，帮助N端结构域正确折叠，并且可以在功能上被 GroESL 替代。