title: High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG.
abstract: BACKGROUND
  For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter. The T7 RNAP drives the expression of the plasmid borne gene encoding the recombinant membrane protein. Production of membrane proteins in the cytoplasmic membrane rather than in inclusion bodies in a misfolded state is usually preferred, but often hampered due to saturation of the capacity of the Sec-translocon, resulting in low yields.
  
  
  RESULTS
  Contrary to expectation we observed that omission of IPTG from BL21(DE3) cells cultured in LB medium can lead to significantly higher membrane protein production yields than when IPTG is added. In the complete absence of IPTG cultures stably produce membrane proteins in the cytoplasmic membrane, whereas upon the addition of IPTG membrane proteins aggregate in the cytoplasm and non-producing clones are selected for. Furthermore, in the absence of IPTG, membrane proteins are produced at a lower rate than in the presence of IPTG. These observations indicate that in the absence of IPTG the Sec-translocon capacity is not/hardly saturated, leading to enhanced membrane protein production yields in the cytoplasmic membrane. Importantly, for more than half of the targets tested the yields obtained using un-induced BL21(DE3) cells were higher than the yields obtained in the widely used membrane protein production strains C41(DE3) and C43(DE3). Since most secretory proteins reach the periplasm via the Sec-translocon, we also monitored the production of three secretory recombinant proteins in the periplasm of BL21(DE3) cells in the presence and absence of IPTG. For all three targets tested omitting IPTG led to the highest production levels in the periplasm.
  
  
  CONCLUSIONS
  Omission of IPTG from BL21(DE3) cells cultured in LB medium provides a very cost- and time effective alternative for the production of membrane and secretory proteins. Therefore, we recommend that this condition is incorporated in membrane- and secretory protein production screens.
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, 106 91, Stockholm, Sweden. zhe.zhang@dbb.su.se.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Xbrane Bioscience AB, 111 45, Stockholm, Sweden. nienke.kuipers@xbrane.com.
authors.forename: Grietje
authors.initials: G
authors.lastname: Kuipers
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, 106 91, Stockholm, Sweden. lukaszniemiec89@gmail.com.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Niemiec
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, 106 91, Stockholm, Sweden. thomas.baumgarten@dbb.su.se.
authors.forename: Thomas
authors.initials: T
authors.lastname: Baumgarten
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands. d.j.slotboom@rug.nl.
authors.forename: Dirk Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, 106 91, Stockholm, Sweden. degier@dbb.su.se.
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: de Gier
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, 106 91, Stockholm, Sweden. anna.hjelm@dbb.su.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Hjelm
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D007544:Isopropyl Thiogalactoside