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@ARTICLE{Mller:897399,
      author       = {Müller, Carolin and Igwe, Chika L. and Wiechert, Wolfgang
                      and Oldiges, Marco},
      title        = {{S}caling production of {GFP}1-10 detector protein in
                      {E}. coli for secretion screening by split {GFP} assay},
      journal      = {Microbial cell factories},
      volume       = {20},
      number       = {1},
      issn         = {1475-2859},
      address      = {London},
      publisher    = {Biomed Central},
      reportid     = {FZJ-2021-03765},
      pages        = {191},
      year         = {2021},
      abstract     = {BackgroundThe split GFP assay is a well-known technology
                      for activity-independent screening of target proteins. A
                      superfolder GFP is split into two non-fluorescent parts,
                      GFP11 which is fused to the target protein and GFP1-10. In
                      the presence of both, GFP1-10 and the GFP11-tag are
                      self-assembled and a functional chromophore is formed.
                      However, it relies on the availability and quality of
                      GFP1-10 detector protein to develop fluorescence by assembly
                      with the GFP11-tag connected to the target protein. GFP1-10
                      detector protein is often produced in small scale shake
                      flask cultivation and purified from inclusion
                      bodies.ResultsThe production of GFP1-10 in inclusion bodies
                      and purification was comprehensively studied based on
                      Escherichia coli as host. Cultivation in complex and defined
                      medium as well as different feed strategies were tested in
                      laboratory-scale bioreactor cultivation and a standardized
                      process was developed providing high quantity of GFP1-10
                      detector protein with suitable quality. Split GFP assay was
                      standardized to obtain robust and reliable assay results
                      from cutinase secretion strains of Corynebacterium
                      glutamicum with Bacillus subtilis Sec signal peptides NprE
                      and Pel. Influencing factors from environmental conditions,
                      such as pH and temperature were thoroughly
                      investigated.ConclusionsGFP1-10 detector protein production
                      could be successfully scaled from shake flask to laboratory
                      scale bioreactor. A single run yielded sufficient material
                      for up to 385 96-well plate screening runs. The application
                      study with cutinase secretory strains showed very high
                      correlation between measured cutinase activity to split GFP
                      fluorescence signal proofing applicability for larger
                      screening studies.},
      cin          = {IBG-1},
      ddc          = {570},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2172 - Utilization of renewable carbon and energy sources
                      and engineering of ecosystem functions (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2172},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {34592997},
      UT           = {WOS:000702398500001},
      doi          = {10.1186/s12934-021-01672-6},
      url          = {https://juser.fz-juelich.de/record/897399},
}