Omitting the inducer IPTG from BL21(DE3) cells cultured in LB medium leads to enhanced production of the membrane proteins YidC and GltP
We routinely use the integral membrane chaperone YidC and the glutamate proton symporter GltP as model membrane proteins to develop cost- and time-effective membrane protein production strategies (e.g., [21]). To facilitate the detection of produced membrane proteins in the cytoplasmic membrane, all target membrane proteins are C-terminally fused to GFP (Fig. 1) [22]. While we were in the process of screening the production of YidC and GltP in BL21(DE3) cells cultured in LB medium, we included as negative controls cultures of BL21(DE3) to which the inducer IPTG was not added. Fluorescence of IPTG induced cultures was monitored 4 and 24 h after the addition of IPTG (Fig. 2a). At the same time-points the fluorescence of the non-IPTG induced cultures was also measured (Fig. 2a). To our surprise, after 24 h, the fluorescence intensities per ml of un-induced cultures were more than five times higher than that of IPTG induced cultures. Also, the A600 values of these cultures were higher than the ones of the IPTG induced cultures (Fig. 2a, Additional file 1: Figure S1).
In all cultures, we monitored GFP fluorescence in individual cells using flow cytometry (Fig. 2b). In the absence of IPTG, cultures producing YidC-GFP and GltP-GFP consisted of a homogenous population of cells, both after 4 and 24 h, and the fluorescence per cell increased over time (Fig. 2b). However, when cells were cultured in the presence of IPTG the cultures consisted of a mixture of producing and non-producing cells, both after 4 and 24 h [21]. The increase of the fraction of non-producing cells over time in IPTG induced cultures indicates that non-producing cells are selected for in the presence of IPTG. This explains why the biomass formation in IPTG induced cultures appears to catch up after 24 h (Fig. 2a). In the presence of IPTG, the fluorescence per cell in the producing population after 4 and 24 h was similar to the fluorescence per cell in the absence of IPTG after 24 h. However, the dramatic increase of the fraction of non-producing cells in the presence of IPTG along with the lower biomass formation results in lower overall production yields.
Thus, when IPTG is omitted from BL21(DE3)/LB medium-based cultures, both YidC-GFP and GltP-GFP appear to be more efficiently produced than when IPTG is added to the cultures.
Characterizing YidC-GFP and GltP-GFP production
To characterize the YidC-GFP and GltP-GFP production process in more detail, we first monitored the integrity of YidC-GFP and GltP-GFP, produced in the cytoplasmic membrane, using in-gel fluorescence [22]. Proteins from whole-cell lysates were separated by SDS-PAGE and subsequently the gel was illuminated with UV light and GFP fluorescence in the gel was captured using a CCD camera (Fig. 3a). For both YidC-GFP and GltP-GFP only one fluorescent band could be detected and they both had the expected molecular weight. The fluorescent bands in lysates of cells cultured in the absence of IPTG were more intense than the ones of cells cultured in the presence of IPTG, which is in keeping with the whole cell fluorescence measurements.
Next, we used an SDS-PAGE/immuno-blotting-based assay that can distinguish between membrane integrated and non-integrated material (see also Fig. 1) [23]. The assay in short: if a membrane protein-GFP fusion is not inserted in the cytoplasmic membrane and ends up in aggregates, its GFP moiety does not fold properly. The GFP moiety folds properly and becomes fluorescent only if the membrane protein-GFP fusion is inserted in the cytoplasmic membrane. Correctly folded GFP is not denatured in SDS-PAGE solubilisation buffer at temperatures below 37 °C. As a consequence, a membrane protein-GFP fusion that has been inserted in the cytoplasmic membrane will migrate faster in a gel than a non-inserted fusion. We monitored the behaviour of both YidC-GFP and GltP-GFP produced in BL21(DE3) in the presence and absence of IPTG using this assay. Through immuno-blotting with an antibody directed against a His-tag, C-terminally attached to GFP, both YidC-GFP and GltP-GFP were detected. In lysates from cells cultured in the presence of IPTG both YidC-GFP and GltP-GFP showed up as two bands: a weak fluorescent one (cytoplasmic membrane integrated) and an intense non-fluorescent one with a higher apparent molecular weight, representing aggregated material in the cytoplasm (Fig. 3b). Most of the produced YidC-GFP and GltP-GFP appeared to end up in aggregates when IPTG was added. When IPTG was omitted from the culture, hardly any non-fluorescent YidC-GFP or GltP-GFP was detected (Fig. 3b). We also monitored the levels of inclusion body protein IbpB, which is a sensitive indicator for the accumulation of aggregated proteins in the cytoplasm [24]. IbpB was clearly present in IPTG induced BL21(DE3) cells and was hardly detectable in non-IPTG induced BL21(DE3) cells (Fig. 3b). These observations are consistent with the hypothesis that non-fluorescent membrane protein GFP fusions accumulate in the cytoplasm upon the addition of IPTG [12]. This observation indicates that upon the addition of IPTG, the production of both YidC-GFP and GltP-GFP leads to saturation of the Sec-translocon capacity. In contrast, in the absence of IPTG the Sec-translocon capacity does not appear to be saturated, resulting in higher yields of membrane proteins produced in the cytoplasmic membrane.
There is a correlation between the rate of membrane protein production and saturation of the Sec-translocon capacity [13]. Therefore, we monitored YidC-GFP and GltP-GFP production over time in BL21(DE3) cells cultured in the presence and absence of IPTG [13] (Fig. 3c). The initial membrane protein production rate in cells cultured in the absence of IPTG was lower than in the presence of IPTG. However, over time more GFP fluorescence, i.e., higher levels of membrane inserted target membrane protein, accumulated in cells cultured in the absence of IPTG than in the presence of IPTG. This result is in keeping with the idea that not adding IPTG leads to a membrane protein production regime that does not saturate the Sec-translocon capacity.
Finally, the produced YidC-GFP and GltP-GFP were characterized in more detail. Cytoplasmic membranes of one liter cultures producing YidC-GFP in the presence and absence of IPTG were isolated. The IPTG induced culture contained 1.3 mg of YidC-GFP per liter and the non-induced culture contained 8.7 mg of YidC-GFP per liter [22]. The total membrane fractions isolated from the IPTG induced culture and from the non-induced culture contained 0.4 and 1.7 mg of YidC-GFP, respectively [22]. Subsequently, the membranes were solubilised in the detergent n-Dodecyl β-d-Maltopyranoside (DDM) and the dispersity of solubilised YidC-GFP was monitored using fluorescence-detection size-exclusion chromatography (FSEC) (Fig. 4a) [25]. YidC-GFP produced in cells both in the presence and absence of IPTG was monodisperse. However, in the absence of IPTG significantly more material was produced. Also cytoplasmic membranes from one liter BL21(DE3)-based cultures producing GltP-GFP in the presence and absence of IPTG were isolated. GltP-GFP was purified and reconstituted in liposomes so that GltP activity (i.e., glutamate uptake) could be monitored. Only membranes isolated from BL21(DE3) cells producing GltP-GFP in the absence of IPTG gave enough material after Immobilized-Metal Affinity Chromatography (IMAC)-based purification to reconstitute GltP-GFP in liposomes and to show that it was active (Fig. 4b) [22]. From a one liter non-induced culture 1.0 mg of GltP-GFP was isolated.
Taken together, omitting IPTG from BL21(DE3)-based cultures greatly increases yields of YidC-GFP and GltP-GFP produced in the cytoplasmic membrane. The produced proteins are of high quality and can be used for further characterization.
Benchmarking the production of membrane proteins in BL21(DE3) cells cultured in LB medium in the absence of IPTG
To benchmark the production of membrane proteins in BL21(DE3) cells cultured in LB medium in the absence of IPTG, we used in addition to YidC-GFP and GltP-GFP six more targets and monitored production of all eight membrane proteins also in the C41(DE3) and C43(DE3) strains (Fig. 5) (Additional file 1: Table S1). Both C41(DE3) and C43(DE3) are widely used to produce membrane proteins [10]. The six additional targets were randomly picked and also fused to GFP at their C-termini. For six out of the eight targets tested, production yields obtained for un-induced BL21(DE3)-based cultures were higher than those obtained for IPTG induced cultures. Importantly, for five out of the eight targets tested BL21(DE3)-based cultures to which no IPTG had been added even outperformed C41(DE3) and C43(DE3).
Taken together, membrane protein production yields using BL21(DE3) cells cultured in LB medium without IPTG are in many instances significantly higher than yields obtained with the established membrane protein production strains C41(DE3) and C43(DE3).
Efficient production of secretory proteins by omitting IPTG to BL21(DE3) cells cultured in LB medium
It has been shown that saturating the Sec-translocon capacity can also hamper the production of secretory proteins in the periplasm [26]. Therefore, we decided to explore the effect of omitting IPTG from BL21(DE3) cultures on the production of secretory Super folder Green Fluorescent Protein (SfGFP), which has a modified DsbA signal sequence at its N-terminus: DsbA*sfGFP [26].
The fluorescence intensities per ml of culture of un-induced BL21(DE3) cells harbouring pETdsbA*sfgfp were significantly higher than the ones of IPTG induced cultures; after 24 h, these values were approximately ten fold higher (Fig. 6a). The amount of biomass formed was negatively affected by IPTG (Fig. 6a). Also the fluorescence intensities per ml of culture obtained for un-induced BL21(DE3) cultures were higher than the ones obtained for C41(DE3) and C43(DE3) based cultures (results not shown). Analysis of BL21(DE3) cells producing SfGFP cultured in the absence and presence of IPTG using fluorescence microscopy resulted in green fluorescent halos which indicates that the SfGFP was efficiently translocated across the membrane to the periplasm (Fig. 6b) [26]. Next, using flow cytometry we showed that cultures producing secretory SfGFP in the absence of IPTG consisted of a homogenous population of cells, both after 4 and 24 h, and that the fluorescence per cell increased over time (Fig. 6c). When cells were cultured for 4 h in the presence of IPTG the fluorescence per cell was significantly higher than in the absence of IPTG, but after 24 h the fluorescence per cell had decreased dramatically and the number of non-producing cells had increased (Fig. 6c). The highest GFP fluorescence intensities, both per ml of culture and per cell, were obtained after 24 h in the absence of IPTG (Fig. 6a, c).
As a control, we also produced SfGFP without a signal sequence in BL21(DE3) in the presence and absence of IPTG. In contrast to secretory SfGFP, cytoplasmic SfGFP was more efficiently produced in the presence of IPTG than in its absence (Fig. 7a). Using flow cytometry experiments showed that addition of IPTG had hardly any negative effect on the amount of SfGFP produced per cell (Fig. 7b), which indicates that the production of SfGFP is indeed not toxic.
We further explored the differences between secretory SfGFP and cytoplasmic SfGFP production by measuring in real-time the accumulation of fluorescence in BL21(DE3)-based cultures, in the presence and absence of IPTG (Fig. 7c). When producing secretory SfGFP in the presence of IPTG, initially fluorescence accumulates rapidly and over time the levels decline. This is most likely due to a negative effect on growth/accumulation of non-producing cells. In contrast, when producing secretory SfGFP in cells in the absence of IPTG, fluorescence accumulates slowly but steadily and at some point exceeds the fluorescence accumulated in cells cultured in the presence of IPTG. In BL21(DE3)-based cultures producing cytoplasmic SfGFP in the presence of IPTG, fluorescence accumulates rapidly and steadily whereas in the absence of IPTG, fluorescence accumulates steadily but only slowly. This indicates that production of SfGFP is not toxic per se, but that translocation of the protein across the cytoplasmic membrane is the critical point. For two more secretory proteins we showed that they were produced more efficiently in BL21(DE3) cells cultured in the absence of IPTG than in the presence of IPTG and that produced proteins were suitable for further experimentation (Additional file 1: Figures S3, S4).
Taken together, omitting IPTG from BL21(DE3) cells cultured in LB medium leads to more efficient production of secretory proteins since the Sec-translocon capacity is not/hardly affected.