| Co-Investigator(Kenkyū-buntansha) |
OKA Masanori Institute of Bioscience Research, Toyobo Co.Senior Researcher, 敦賀バイオ研究所, 主席部員
KURIHARA Tatsuo Institute for Chemical Research, Kyoto University Instructor, 化学研究所, 助手 (70243087)
YOSHIMURA Tohru Institute for Chemical Research, Kyoto University Instructor, 化学研究所, 助手 (70182821)
SODA Kenji Institute for Chemical Research, Kyoto University Professor, 化学研究所, 教授 (30027023)
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| Budget Amount *help |
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
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
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