Özet:
The gene encoding the Taq I restriction endonuclease was cloned into pMAL-c2 plasmid to construct p22 expressing the MBP-Taq I fusion protein. The affinity of MBP to amylose was exploited for the single-step chromatographic purification. To protect the Taq I endonuclease gene from the cleavage by its own product, the co-expression of Taq I methylase was recommended. Therefore, the gene encoding the Taq I methylase was PCR amplified and a recombinant plasmid (pETMET) expressing this enzyme was constructed. In this study E. coli cells were transformed by p22 and pETMET. The optimum host strain, induction time and period using this system was determined by shake-flask experiments. The highest productivity (5.105 U/L Taq I endonuclease activity) was obtained when the recombinant E. coli TB1 (p22, pETMET) cells were grown in LB medium and induced at the early exponential phase of the growth for 10 hours. The growth, plasmid stability, and Taq I endonuclease activity were investigated under fully controlled operating conditions using a fermentor. The maximum total Taq I endonuclease activity (7,5.105 U/L) could be reached when the recombinant cells were induced for 12 hours in SB Medium. In conclusion, the presence of pETMET has slightly improved the yield of MBP-Taq I endonuclease production.
