Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress

Abstract

In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Keywords: Escherichia coli; Sec translocon; periplasm; proteomics; recombinant protein production.

FIG 1

Periplasmic production of the scFv BL1 under nonoptimized and optimized conditions. Production of the scFv BL1 was induced with IPTG in Tuner(DE3) cells harboring pLemo in the absence and presence of 500 μM l-rhamnose. Tuner(DE3) cells harboring pLemo and an empty expression vector cultured in the presence of 500 μM l-rhamnose were used as a control. (A) Four hours after induction, biomass formation was monitored by measuring A₆₀₀. (B, top) Levels of the precursor form (p) and the mature form (m) of the scFv BL1 were monitored using SDS-PAGE followed by immunoblotting in whole cells cultured in the absence and presence of 500 μM l-rhamnose using an α-His antibody recognizing the C-terminal His tag of the scFv BL1. Cells were converted into spheroplasts and the periplasmic fractions were isolated. Levels of the scFv BL1 were also monitored in the periplasmic fractions and spheroplasts. The periplasmic protein SurA and the cytoplasmic protein GroEL were used as markers to monitor the spheroplasting efficiency and the isolation of the periplasmic fractions. (B, bottom) PVDF membranes containing decreasing amounts of β-galactosidase were incubated with whole-cell lysate. Binding of the scFv BL1 present in whole-cell lysates to the β-galactosidase was detected using an α-His antibody recognizing the C-terminal His tag of the scFv BL1.

FIG 2

Consequences of the nonoptimized production of the scFv BL1. Production of the scFv BL1 was induced with IPTG in Tuner(DE3) cells harboring pLemo in the absence of l-rhamnose. Tuner(DE3) cells harboring pLemo and an empty expression vector were used as a reference. The proteomes of aforementioned cells were analyzed using a combination of label-free mass spectrometry and immunoblotting. (A) Mass spectrometry was used to determine fold changes in the abundance of T7 RNAP and proteins involved in transcription, protein synthesis, the TCA cycle, the Pta pathway, and protein quality control. Fold changes are plotted as log2 (fold change) on the y axis (see Table S2 in the supplemental material). (B, left) Accumulation levels of the σ³²-regulated proteins IbpB, DnaK, GroEL, and FtsH along with σ³² itself were monitored by immunoblotting. (B, middle) The presence of the precursor form (p) and mature form (m) of OmpA was probed by immunoblotting. (B, right) The accumulation levels of the Sec translocon components SecY, SecE, SecA, and YidC were monitored by immunoblotting as well.

FIG 3

Consequences of optimizing the production of the scFv BL1 using the Lemo setup. The scFv BL1 was produced in Tuner(DE3) cells harboring pLemo cultured in the presence of 500 μM l-rhamnose and the absence of l-rhamnose (see Fig. 1). Tuner(DE3) cells harboring pLemo and an empty expression vector cultured in the presence of 500 μM l-rhamnose were used as a reference (see Fig. 1). The proteome composition of cells was analyzed using label-free mass spectrometry. The q values, plotted as −log10 on the y axis, are plotted against the relative fold changes in protein abundance, plotted as log2 on the x axis (see Table S5 in the supplemental material). The horizontal dashed line indicates the significance threshold; q values below the line represent changes that are not considered to be significant, whereas q values above the line represent changes that are considered to be significant. , proteins in cells producing the scFv BL1 under optimized conditions; , proteins in cells producing the scFv BL1 under nonoptimized conditions.

FIG 4

Release of the scFv BL1 in the extracellular medium. The scFv BL1 was produced in Tuner(DE3) cells harboring pLemo cultured in the presence of 500 μM l-rhamnose. The culture medium was cleared from whole cells by centrifugation followed by filtration. The OMVs in the spent medium were subsequently isolated by ultracentrifugation. (A) Levels of the scFv BL1, the cytoplasmic markers GroEL and σ⁷⁰, the outer membrane marker OmpA, and the periplasmic marker SurA were monitored in whole cells, the spent medium, and isolated OMVs using SDS-PAGE followed by immunoblotting. The scFv BL1 was detected using an α-His antibody recognizing the C-terminal His tag of the scFv BL1. (B) The localization of the scFv BL1 associated with OMVs was examined using a proteinase K protection assay. OMVs were treated with either Triton X-100 (left lane), Triton X-100 and proteinase K (middle lane), or proteinase K only (right lane). Samples were analyzed using SDS-PAGE followed by immunoblotting and the scFv BL1 was detected using an α-His antibody recognizing the C-terminal His tag of the scFv BL1. (C) The proper folding of the scFv BL1 was assessed using a dot blot assay. Decreasing concentrations of β-galactosidase were spotted onto a PVDF membrane and incubated with Triton X-100-lysed OMVs. The scFv BL1 bound to the β-galactosidase was detected using an α-His antibody recognizing the C-terminal His tag of the scFv BL1.