Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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GroEL/ES Chaperone and Low Culture Temperature Synergistically Enhanced the Soluble Expression of CGTase in E. coli

  • Park, So-Lim (Department of Biotechnology & Bioengineering, Dong-Eui University) ;
  • Kwon, Mi-Jung (Department of Biotechnology & Bioengineering, Pukyong National University) ;
  • Kim, Sung-Koo (Neo Pharm, BVC 307, KRIBB) ;
  • Nam, Soo-Wan (Department of Biotechnology & Bioengineering, Pukyong National University)
  • Published : 2004.02.01
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Abstract

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.

Keywords

References

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