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KMID : 0613820040140010121
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Journal of Life Science
2004 Volume.14 No. 1 p.121 ~ p.125
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Enhancement of Soluble Expression of CGTase in E. coli By Chaperone Molecules and Low Temperature Cultivation
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Park So-Lim
Kim Sung-Koo
Nam Soo-Wan
Kwon Mi-Jung
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Abstract
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The synergistic effect of lowered incubation temperature and GroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGT1 and pGro11 carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-1 promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at 25℃ with groEL/ES expression improved the activity of CGTase by two fold, compared to 37℃ cultivation without chaperones. SDS-PAGE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at 25℃, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at 37℃ without chaperone. The amount of soluble CGTase from 25℃ culture with chaperone was 3.5-fold higher than that of 37℃ culture without chaperone. Therefore the expression of GroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.
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KEYWORD
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low temperature cultivation, CGTase, GroEL/ES, Escherichia col
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