Application of Molecular Chaperone to Protein Engineering

过量生产的重组蛋白通常在宿主细胞中以包涵体的形式获得，并且有时难以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基因的共表达引起大肠杆菌中产生的人胶原原酶的溶解性增加。本研究的目的是建立一种通过共产生分子伴侣来防止重组蛋白形成包涵体的方法。过表达的murI（glr）基因，它编码的谷氨酸消旋酶的大肠杆菌，导致在形成的包涵体的酶，和小的活动被发现在可溶性馏分的大肠杆菌细胞。groESL基因与murI的共表达引起谷氨酸消旋酶的活性形式在体内溶解。谷氨酸消旋酶的可溶性取决于GroESL g表达的时间和量 ...更多信息 恩。在这项研究中，我们还研究了GroESL对细菌丙氨酸消旋酶结构域肽折叠的影响。嗜热脂肪芽孢杆菌耐热丙氨酸消旋酶的一个亚基是由两个结构域组成的同源二聚体蛋白。当编码对应于结构域的N-和C-末端肽片段的基因在相同的宿主细胞中串联或单独表达时，产生活性片段酶。然而，当单独表达N端或C端片段基因时，含有赖氨酸39与辅因子吡哆醛5 '-磷酸结合的N端片段大多仅以不溶性形式产生，而很少发现C端片段。可溶性N端片段与分子伴侣GroESL共产生，并显示丙氨酸消旋酶活性。变性后的N端片段恢复了GroESL的复性活性，因此只有N端结构域参与催化作用，C端结构域作为分子内伴侣帮助N端结构域正确折叠，并可被GroESL功能性取代。少

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