Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein production. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E. coli RNase E, RraA. E. coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, respectively, accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of commercial strains frequently utilized for recombinant membrane protein production purposes.

Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon. Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids. The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer. Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively. Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC. For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255. The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218. Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC. This IncC-KorB contact differs in location from the ParA-ParB/SopA-SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant.

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The nicotinic acetylcholine receptor (AChR) is a member of the superfamily of ligand-gated ion channels, which also includes the glycine, c-aminobutyric acid A, and 5-HT 3 receptors [1]. Its physiological role is to mediate the fast chemical transmission of electrical signals in response to acetylcholine released from the nerve terminal to the end-plate.The muscle AChR is a transmembrane glycoprotein ( 290 kDa) located on the postsynaptic membrane of the neuromuscular junction and is composed of five The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (a 2 bcd or a 2 bce), is the major antigen in the autoimmune disease, myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and inactivate, the AChR. The extracellular domain (ECD) of the human muscle a subunit has been heterologously expressed and extensively studied.Our aim was to obtain satisfactory amounts of the ECDs of the non-a subunits of human muscle AChR for use as starting material for the determination of the 3D structure of the receptor ECDs and for the characterization of the specificities of antibodies in sera from patients with MG. We expressed the N-terminal ECDs of the b (amino acids 1-221; b1-221), c (amino acids 1-218; c1-218), and e (amino acids 1-219; e1-219) subunits of human muscle AChR in the yeast, Pichia pastoris. b1-221 was expressed at 2 mgAEL )1 culture, whereas c1-218 and e1-219 were expressed at 0.3-0.8 mgAEL )1 culture. All three recombinant polypeptides were glycosylated and soluble; b1-221 was mainly in an apparently dimeric form, whereas c1-218 and e1-219 formed soluble oligomers. CD studies of b1-221 suggested that it has considerable b-sheet secondary structure with a proportion of a-helix. Conformation-dependent mAbs against the ECDs of the b or c subunits specifically recognized b1-221 or c1-218, respectively, and polyclonal rabbit antiserum raised against purified b1-221 bound to 125 I-labeled a-bungarotoxin-labeled human AChR. Moreover, immobilization of each ECD on Sepharose beads and incubation of the ECD-Sepharose matrices with MG sera caused a significant reduction in the concentrations of autoantibodies in the sera, showing specific binding to the recombinant ECDs. These results suggest that the expressed proteins present some near-native conformational features and are thus suitable for our purposes.Abbreviations AChR, nicotinic acetylcholine receptor; ECD, extracellular domain; MG, myasthenia gravis; b1-221, amino acids 1-221 of the human AChR b subunit; c1-218, amino acids 1-218 of the human AChR c subunit; e1-219, amino acids 1-219 of the human AChR e subunit.

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