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Expression of a thermostable restriction endonuclease in recombinant Escherichia coli cells and optimization of fermentation conditions

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Abstract

Taq I restriction endonuclease gene of the thermophilic eubacterium Thermus aquaticus YT-1 (ATCC 25104) was successfully cloned and expressed in recombinant Escherichia coli cells under the control of the lac promoter/operator system. Higher Taq I endonuclease specific activities and biomass yields were obtained from E. coli ER2508(pUCTaq) cells when they were induced at the late-exponential phase of their growth. Taq I endonuclease expression was found to be host strain-dependent such that, among the three different strains examined, E. coli XL1(pUCTaq) produced the highest specific Taq I endonuclease activities for longer induction periods. Decreasing the inducer concentration from 1 to 0.1 mM not only improved the specific enzyme activity yields but also is more economical, considering the high cost of isopropyl-β-D-thiogalactopyranoside (IPTG). The optimum culture temperature was found to be 37 °C. Taq I endonuclease specific activity recovered from E. coli XL1(pUCTaq) cells was 935 U/mg under optimum conditions.

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Authors and Affiliations

  1. Department of Chemical Engineering, Boğaziçi University, 80815, Bebek, Istanbul, Turkey

    E. Toksoy, Z.İ. Önsan & B. Kırdar

Authors
  1. E. Toksoy
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  2. Z.İ. Önsan
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  3. B. Kırdar
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Toksoy, E., Önsan, Z. & Kırdar, B. Expression of a thermostable restriction endonuclease in recombinant Escherichia coli cells and optimization of fermentation conditions. World Journal of Microbiology and Biotechnology 18, 23–27 (2002). https://doi.org/10.1023/A:1013921713072

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  • DOI: https://doi.org/10.1023/A:1013921713072

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