High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, cilt.59, sa.2-3, ss.239-245, 2002 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 59 Sayı: 2-3
- Basım Tarihi: 2002
- Doi Numarası: 10.1007/s00253-002-1001-0
- Dergi Adı: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
- Sayfa Sayıları: ss.239-245
- Marmara Üniversitesi Adresli: Evet
Özet
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 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.