High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG

Abstract

BackgroundFor 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.ResultsContrary 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.ConclusionsOmission 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0328-z) contains supplementary material, which is available to authorized users.

Induction of the pneumococcal vncRS operon by lactoferrin is essential for pneumonia

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“…For online GFP fluorescence measurements 200 μl of cultures were transferred after induction with IPTG at an OD 600 of ~0.4 to a 96 well plate and fluorescence was automatically detected every 5 minutes. The 96 well plate was shaken every 30 seconds36.…”

“…Production of membrane protein GFP fusions was monitored 24 hours after the addition of IPTG using whole-cell fluorescence as described before36. Standard deviations are based on a minimum of three biologically independent experiments.…”

“…Standard deviations are based on a minimum of three biologically independent experiments. GFP fluorescence was analyzed on a single cell level by flow cytometry using a FACSCalibur instrument (BD Biosciences) as described before36. FM4-64 membrane staining was used to discriminate between cells and background signal.…”

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