% IMPORTANT: The following is UTF-8 encoded.  This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.

@ARTICLE{Bakkes:887941,
      author       = {Bakkes, Patrick J. and Ramp, Paul and Bida, Astrid and
                      Dohmen-Olma, Doris and Bott, Michael and Freudl, Roland},
      title        = {{I}mproved p{EKE}x2-derived expression vectors for tightly
                      controlled production of recombinant proteins in
                      {C}orynebacterium glutamicum},
      journal      = {Plasmid},
      volume       = {112},
      issn         = {0147-619X},
      address      = {Orlando, Fla.},
      publisher    = {Academic Press},
      reportid     = {FZJ-2020-04533},
      pages        = {102540 -},
      year         = {2020},
      note         = {Biotechnologie 1},
      abstract     = {The Escherichia coli/Corynebacterium glutamicum shuttle
                      vector pEKEx2 is an IPTG-inducible expression vector that
                      has been used successfully for the synthesis of numerous
                      proteins in C. glutamicum. We discovered that the leaky gene
                      expression observed for pEKEx2-derived plasmids relates to
                      reduced functionality of the plasmid-encoded repressor LacI
                      carrying a modified C-terminus, while duplicate DNA
                      sequences in the pEKEx2 backbone contribute to plasmid
                      instability. We constructed the pEKEx2-derivatives pPBEx2
                      and pPREx2, which harbor a restored lacI gene and which lack
                      the unnecessary duplicate DNA sequences. pPREx2 in addition
                      enables fusion of target genes to a C-terminal Strep-tag II
                      coding region for easy protein detection and purification.
                      In the absence of inducer, the novel vectors exhibit tight
                      gene repression in C. glutamicum, as shown for the secretory
                      production of Fusarium solani pisi cutinase and the
                      cytosolic production of green fluorescent protein and C.
                      glutamicum myo-inositol dehydrogenase. Undesired
                      heterogeneity amongst clones expressing cutinase from pEKEx2
                      was attributed to the loss of a vector fragment containing
                      the cutinase gene, which likely occurred via homologous
                      recombination of the identical flanking DNA sequences. Such
                      loss was not observed for pPBEx2. Using pPREx2, IolG-Strep
                      was successfully produced and purified to homogeneity by
                      Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG
                      with a specific activity of 27 μmol·min−1·(mg
                      protein)−1 from 100 mL culture. The tight gene repression
                      in the absence of inducer and the improved plasmid stability
                      make expression vectors pPBEx2/pPREx2 attractive
                      alternatives to the available molecular tools for genetic
                      manipulation and high-level production of recombinant
                      proteins in C. glutamicum.},
      cin          = {IBG-1},
      ddc          = {570},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {581 - Biotechnology (POF3-581)},
      pid          = {G:(DE-HGF)POF3-581},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {32991924},
      UT           = {WOS:000598058600002},
      doi          = {10.1016/j.plasmid.2020.102540},
      url          = {https://juser.fz-juelich.de/record/887941},
}