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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Background: PARP13 contains a divergent PARP homology ADP-ribosyltransferase domain of unknown function. Results: The consensus NAD ϩ pocket of PARP13 is occluded by interacting protein side chains.

It has been assumed that CODHII Ch catalyzes the oxidation of CO at the Ni-( 2 S)-Fe1 subsite of cluster C (3). The prime candidate for CO binding is the nickel ion because of its facile accessibility through the substrate channel and its empty apical coordination site (3). Fe1 is the presumed OH Ϫ donor ligand in CO 2 formation (3, 6). The CODH Oc from the aerobic bacterium Oligotropha carboxidovorans oxidizes CO at the Mo-( 2 S)-Cu subsite of the [Cu-S-MoO 2 ] active site, in which copper and molybdenum are bridged by a cyanolyzable sulfane 2 S (7, 8). The enzyme is inactivated when 2 S is removed and reactivated when 2 S is reinserted (9). The Mo-( 2 S)-Cu subsite resembles the Ni-( 2 S)-Fe1 bridge in cluster C of CODHII Ch . The mechanism of CO oxidation based on the x-ray structure of [Cu-S-MoO 2 ] CODH Oc with bound inhibitor n-butyl isocyanide involves a thiocarbonate-like intermediate state and proposes the binding of CO between the 2 S and copper (equivalent to nickel in CODHII Ch ) and the binding of an OH Ϫ group at molybdenum (equivalent to Fe1 in CODHII Ch ) (7).Structures of cluster C of Ni-Fe CODHs from Rhodospirillum rubrum (CODH Rr ) (10) and Moorella thermoacetica (CODH Mt ) (11, 12) also showed the positions of the five metal ions in cluster C of CODHII Ch but did not reveal the bridging 2 S. Since sodium sulfide was found to inhibit CODH Rr and CODH Mt , it has been concluded that cluster C with the bridging 2 S, as has been observed in CODHII Ch from C. hydrogenoformans (3, 4), might represent an inhibited form (13). On the other hand, it has been shown that the [Ni-4Fe-4S] cluster miss-

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