A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used ''Walker strains'' C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for highthroughput applications.engineering ͉ systems biotechnology ͉ proteomics T he natural abundance of membrane proteins is typically too low to isolate sufficient amounts of material for functional and structural studies. Therefore, membrane proteins must be obtained by overexpression, and the bacterium E. coli is the most widely used vehicle for this purpose (1). Although many membrane proteins can be overexpressed in inclusion bodies, their refolding into functional proteins is often not successful (2). To avoid the refolding problem, overexpression of membrane proteins by accumulation in the cytoplasmic membrane is needed. However, overexpression is often toxic to the cell, thereby preventing biomass formation and severely reducing yields (1). Thus, membrane protein overexpression has to be optimized, but no systematic, generic, and high-throughput-compatible method is available for the optimization process.Bacteriophage T7 RNA polymerase (T7RNAP) is often used to drive recombinant protein production in E. coli (3). In BL21(DE3) and its derivatives, the gene encoding T7RNAP is under control of the lacUV5 promoter, a strong variant of the wild-type lac promoter. It is insensitive to catabolite repression and, therefore, controlled only by the lac repressor, LacI, which binds to the lac operator (4). T7RNAP exclusively recognizes the T7 promoter and it transcribes eight times faster than E. coli RNAP allowing high yield protein production (5). Most T7 expression vectors employ a T7lac hybrid promoter that combines the strong T7 10 promoter with a lac operator to diminish leaky expression. On addition of the inducer isopropyl ␤-Dthiogalactoside (IPTG), lacI repression is relieved, resulting in recombinant protein production. If toxicity due to leaky expression is a problem, T7RNAP activity can be further dampened with the T7RNAP inhibit...

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BackgroundIn Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled.ResultsBoth SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized.ConclusionsSaturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment.