Consequences of Membrane Protein Overexpression in Escherichia coli*□S

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 oneand 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. Molecular & Cellular Proteomics 6:1527–1550, 2007. In both proand eukaryotes 20–30% of all genes encode -helical transmembrane domain (TMD) proteins, which act in various and often essential capacities (1, 2). Notably these TMD proteins (hereafter referred to as membrane proteins) play key roles in disease, and they constitute more than half of all known drug targets (e.g. Ref. 3). The natural abundance of membrane proteins is in general too low to conveniently isolate sufficient material for functional and structural studies (4, 5). Therefore, membrane proteins are often obtained through overexpression. The bacterium Escherichia coli is the most widely used vehicle for this purpose with overexpressed proteins accumulating in the cytoplasmic membrane (also named inner membrane) or in cytoplasmic inclusion bodies (4). Although membrane proteins can often more easily be expressed in inclusion bodies, their refolding into functional proteins is challenging and often not successful (6). Overexpression of membrane proteins through accumulation in a membrane system avoids this refolding problem but is usually toxic to the organism, thereby severely reducing yields (4). The reasons for this toxicity are not clear; therefore, a better understanding of the physiological response to overexpression is needed to improve such yields through rational design (e.g. through engineering of strains or modifying target proteins). Because optimal protein production conditions cannot be predicted, yield maximization is currently mostly done by “trial and error.” However, green fluorescent protein (GFP)-based methodology developed for E. coli now facilitates rapid screening for overexpression in the cytoplasmic membrane and can accelerate the trial and error process (7, 8). Improved prediction of protein overexpression success would be very beneficial but requires an understanding of the physiological response of the cell to overexpression. It is generally assumed that the overexpressed membrane protein affects integrity of the membrane and thus cell viability, leading to e.g. reduced growth and hampered division (4). In addition, overexpression of membrane proteins may lead to