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 down-regulated 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.

The construction and characterization of a combinatorial library of a solvent-exposed surface of an α-helical domain derived from a bacterial receptor is described. Using a novel solid-phase approach, the library was assembled in a directed and successive manner utilizing single-stranded oligonucleotides containing multiple random substitutions for the variegated segments of the gene fragment The simultaneous substitution of 13 residues to all 20 possible amino acids was carried out in a region spanning 81 nucleotides. The randomization was made in codons for amino acids that were modelled to be solvent accessible at a surface made up from two of the three a-helices of a monovalent Fc-binding domain of staphylococcal protein A. After cloning of the PCR-amplified library into a phagemid vector adapted for phage display of the mutants, DNA sequencing analysis suggested a random distribution of codons in the mutagenized positions. Four members of the library with multiple substitutions were produced in Escherichia coli as fusions to an albumin-binding affinity tag derived from streptococcal protein G. The fusion proteins were purified by human serum albumin affinity chromatography and subsequently characterized by SDSelectrophoresis, CD spectroscopy and biosensor analysis. The analyses showed that the mutant protein A derivatives could all be secreted as soluble full-length proteins. Furthermore, the CD analysis showed that all mutants, except one with a proline introduced into helix 2, have secondary structures in close agreement with the wild-type domain. These results proved that members of this α-helical receptor library with multiple substitutions in the solvent-exposed surface remain stable and soluble in E.coli. The possibility of using this library for a phenotypic selection strategy to obtain artificial antibodies with novel functions is discussed.