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KMID : 0613820020120060688
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Journal of Life Science
2002 Volume.12 No. 6 p.688 ~ p.693
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Improvement of production of active cyclodextrin glucanotransferase by coexpression GroEL/ES chaperone in E. coli
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Kwon Mi-Jung
Park So-Lim
Kim Byung-Woo
Kim Sung-Koo
Nam Soo-Wan
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Abstract
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Molecular chaperones prevent the misfolding of newly synthesized polypeptides in the cell. The coexpression of molecular chaperones could be expected to improve the production of soluble and active recombinant proteins. In this study, the effect of coexpression of E. coli GroEL/ES chaperone on the active production of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in E. coli was investigated. Two plasmids, pTCGT1 and pGro7 in which the cgt and the groEL/ES genes are under the control of T7 promoter and araB promoter, respectively, were co-transformed into E. coli. With a series of cultures of recombinant E. coli cells, the optimal concentrations of IPTG and L-arabinose were found be 1 mM and 0.3 mg/ml, respectively. When IPTG and L-arabinose were added at 0.8～1.0 OD600 and 0.4～0.5 OD600, active CGTase production was increased significantly. This coexpression condition resulted in 1.5-fold increased level of soluble CGTase (0.7～0.73 unit/ml), compared to the level of CGTase in the single expression (0.36～0.56 unit/ml). An SDS-PAGE analysis revealed that about 33.6% of CGTase in the total CGTase protein was found in the soluble fraction by coexpression of GroEL/ES chaperone.
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KEYWORD
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Coexpression, cyclodextrin glucanotransferase, GroEL/ES chaperone, Escherichia coli
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