High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter.
Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia colicells. 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 250x10(6) U l(-1) and 350x10(6) 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.
Department of Chemical Engineering, Bogaziçi University, Bebek, 80815 Istanbul, Turkey.
E
E
Toksoy
Z I
ZI
Onsan
B
B
Kirdar
D017123:Bacteriophage T7 / Q000235:genetics*; D019149:Bioreactors; D003470:Culture Media; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D005285:Fermentation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*
D003470:Culture Media; D011994:Recombinant Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases