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

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--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.