Abstract.
Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia coli cells. In system [R], the TaqI endonuclease gene was cloned and expressed under the control of the strong T7 RNA polymerase promoter. To protect cellular DNA, methylase protection was provided by constitutive co-expression of TaqI methylase activity either by cloning the TaqI methylase gene on a second plasmid (system [R,M]) or by constructing a recombinant plasmid harboring both the endonuclease and methylase genes (system [R+M]). In batch shake flasks containing complex media, co-expression of the methylase gene in systems [R,M] and [R+M] resulted in a 2- and 3-fold increase in volumetric productivity over system [R], yielding activities of 250×106 U l–1 and 350×106 U l–1, which were 28 and 39 times higher than the data in the literature, respectively. Under controlled bioreactor conditions in chemically defined medium, co-expression of methylase activity greatly improved the yield and specific TaqI endonuclease productivity of the recombinant cells, and reduced acetic acid excretion levels. System [R,M] is preferable for high expression levels at longer operation periods, while system [R+M] is well-suited for high expression levels in short-term bioreactor operation.
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Toksoy, .E., Önsan, .Z. & Kirdar, .B. High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter. Appl Microbiol Biotechnol 59, 239–245 (2002). https://doi.org/10.1007/s00253-002-1001-0
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DOI: https://doi.org/10.1007/s00253-002-1001-0