Application of Molecular Chaperone to Protein Engineering

大量生产的重组蛋白通常是以包涵体的形式在宿主细胞中获得的，有时在体外很难将包涵体溶解成活性形式。最近，有报道称，分子伴侣基因dnaK和groESL的共表达导致了人原胶原酶在大肠杆菌中产生的可溶性增加。本研究的目的是建立一种通过分子伴侣的共同产生来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)基因的过表达导致了该酶的包涵体的形成，转化细胞中的可溶性部分几乎没有活性。GroESL基因与Muri的共表达导致了谷氨酸消旋酶以活性形式在体内的增溶。谷氨酸消旋酶的溶解性取决于GroESL g…表达的时间和数量更多的烯。在这项研究中，我们还检测了GroESL对细菌丙氨酸外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外消旋酶亚基是一种同源二聚体蛋白，由两个结构域组成。当编码相应结构域的N-末端和C-末端多肽片段的基因在同一宿主细胞中串联或单独表达时，就产生了活性片段酶。然而，当单独表达N端片段或C端片段时，含有与辅因子吡哆醛5‘-磷酸结合的赖氨酸39的N端片段大多只以不溶的形式产生，而C端片段很少。可溶性N-末端片段与分子伴侣GroESL共同产生，并显示丙氨酸消旋酶活性。变性的N-末端片段恢复了GroESL的复性活性，因此，只有N-末端区域参与催化，而C-末端片段作为一种分子内伴侣，帮助N-末端片段正确折叠，并可被GroESL功能取代。较少

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