Consequences of Membrane Protein Overexpression in Escherichia coli

Abstract

Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one-and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/ S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a downregulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

“…We found that in E. coli this gene presents an iTSS (TSS_873, locus b0733) for a putative intraRNA that, if translated using the first available ATG start codon 29 nt downstream, would produce a 43.9 kDa isoform of the 58.2 kDa CydA cognate protein. Mass spectrometry identification of spots in 2D gels of cytoplasmic membranes performed ten years ago [70] showed CydA (among others) spots with observed MW incompatible with full-length protein but consistent with isoform. Contrary to previous examples, this protein is not highly acidic and do not meet the criteria for MW correction.…”

Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

“…However, the concentrations of E,Z-PAOx and PAN in the culture broths of the ATCC 31882 transformants were far lower than those of the C41(DE3) transformants, probably due to the lower biomass concentrations of the ATCC 31882 transformants than the C41 transformants. These negative results for the ATCC 31882 transformants can be explained by its lower tolerance of heterologous expression of the membrane proteins than the C41 strain (42). Thus, it was suggested that in addition to the ability to biosynthesize L-Phe, the tolerance of a host cell for heterologous expression of membrane protein has to be improved to increase the concentrations of E,Z-PAOx and PAN.…”

Autotransporter-Based Antigen Display in Bacterial Ghosts

“…1C). Both the forward and side scatters of ghosts and control cells were monitored, which allowed comparing their size and internal complexity, respectively (32)(33)(34). The size (forward scatter) of a large fraction of the ghosts resembled the size of the control cells (Fig.…”

“…EC452 was cultured as described above; IPTG was added to the culture at an OD 600 of 0.25 for induction of gfp expression (see "Strains, plasmids, and culture conditions" above) and E gene expression was subsequently induced at an OD 600 of 0.5 by the addition of 6 mM L-rhamnose. EC452 cells/BGs were harvested immediately before lysis induction and 120 min after induction, treated as described above, and stained with the membrane dye FM4-64 as described before (32). Prior to imaging, cells were placed on a microscope glass slide coated with a premade agarose pad (1% [wt/vol] agarose) and were left to immobilize for ϳ5 min.…”

“…Cell/BG size (forward scatter), granularity (side scatter), and fluorescence (FM4-64, cytoplasmic GFP, and Oregon Green 488 probe of the secondary antibody used for ESAT6 labeling) were analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences) essentially as described before (32). Cells/BGs were prepared as described under "Fluorescence microscopy."…”