Tuning Escherichia coli for membrane protein overexpression

Abstract

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...

Genetic selection system for improving recombinant membrane protein expression in E. coli

“…These mutant strains were recently shown to act by slowing expression from the strong T7 promoter. 7 These results clearly indicate that it is possible to reengineer E. coli with an improved ability to express membrane proteins.…”

“…For example, the C41 and C43 effects were found to be specific to the T7 promoter. 7 We therefore lysogenized all strains with kDE3 to introduce T7 RNA polymerase and tested the mutants for efficacy in a T7 promoter system. As shown in Figure 5, all of the mutants improved expression of Rv1337 behind the T7 promoter up to fourfold.…”

“…It is likely that the reduced copy number in EXP-Rv1337-4 slows down expression to a level that the cells can more readily accommodate, much like the C41 and C43 strains do for expression from the T7 promoter. 7,13 …”

“…Wagner et al found that membrane protein expression could be improved considerably by fine tuning induction levels. 7 In addition, fusion to other proteins can help expression, and fusion to Mistic appears to be particularly helpful and is thought to help chaperone the protein into the membrane. 8 Also, the membrane protein itself can be mutated to generate more stable variants that express at higher levels, but the disadvantage is that the product may not reflect the native structure and function.…”

Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

“…For example, the widely-used E. coli "Walker strains" (C41 and C43) apparently result in higher yields of recombinant membrane protein because their growth is not strongly inhibited upon induction. This results in increased biomass rather than increased "per cell" yields [25]. In yeast, however, the specific activity of some GPCRs is often lower when high cell density cultures are induced [26].…”