High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters fast bioprocess development is much more challenging, especially heterologous protein production. Here, the nature of foreign makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature substrate feed rate...

Pulse-chase experiments were performed to follow the export of Escherichia coli outer membrane protein OmpA.Besides pro-OmpA protein, which carries a 21-residue signal sequence, three species ompA gene products distinguishable.One probably represented an incomplete nascent chain, another mature in membrane, and third, designated imp-OmpA (immature processed), was already processed but apparently still associated with plasma membrane.The pro-and proteins could be characterized more fully by...

Technical bulk enzymes represent a huge market, and the extracellular production of such is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary direct target protein into translocation machinery. For example, >150 Sec-specific SPs have been identified Bacillus subtilis alone. As best SP choice cannot predicted priori, screening homologous has shown powerful tool different...

The periplasmic, NADP‐containing glucose‐fructose oxidoreductase of the gram‐negative bacterium Zymomonas mobilis belongs to a class redox cofactor‐dependent enzymes which are exported with aid signal peptide containing so‐called twin‐arginine motif. In this paper we show that replacement one or both arginine residues results in drastically reduced translocation periplasm, showing motif is essential. Mutant proteins which, contrast wild‐type oxidoreductase, bind NADP looser and dissociable...

The twin-arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates the transmembrane transport folded proteins, which harbour signal sequences with a conserved motif. Many Tat translocases comprise three membrane proteins TatA, TatB TatC. TatC was previously shown to be involved in recognizing peptides. Here we show that beyond recognition, insertion sequence, thereby translocating sequence cleavage site across bilayer. In absence TatB, this can lead removal even from...

In Escherichia coli, the Tat system promotes membrane translocation of a subset exported proteins across cytoplasmic membrane. Four genes (tatA, tatB, tatC, and tatE) have been identified that encode components E. coli apparatus. Whereas TatA TatE can functionally substitute for each other, TatB TatC shown to perform distinct functions. contrast systems ABC(E) type found in many other bacteria, some microorganisms possess TatAC-type translocase consists only, suggesting that, these systems,...

Abstract A series of overlapping deletions has been constructed in the ompA gene which encodes 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that OmpA fragments were either located periplasmic space or associated with membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing found to be periplasmic. The nature association remained unknown; no membrane-protected tryptic could identified any...

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across cytoplasmic membrane bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive residues that are thought to be involved in recognition by Tat translocase. Here, we have analyzed specificity peptide using genetic approach. Replacement precursor protein lysine-glutamine resulted an export-defective mutant was no longer...

The ompA gene from Salmonella typhimurium, encoding a major heat-modifiable protein of the outer membrane, has been cloned and extensively characterized. When expressed in Escherichia coli directs synthesis an OmpA which is functionally topologically indistinguishable that made S. thus indicating export membrane incorporation are very similar two organisms. typhimurium effectively substitutes for E. polypeptide F-dependent conjugation uptake certain colicins, although it cannot serve as...

Signal peptides direct the export of secretory proteins from cytoplasm. After processing by signal peptidase, they are degraded in membrane and The resulting fragments can have signaling functions. These observations suggest important roles for peptide peptidases. present studies show that Gram-positive eubacterium Bacillus subtilis contains two genes proteins, denoted SppA TepA, with similarity to peptidase A Escherichia coli. Notably, TepA also shows ClpP proteases. B. was only required...

In Escherichia coli, the SecB/SecA branch of Sec pathway and twin-arginine translocation (Tat) represent two alternative possibilities for posttranslational proteins across cytoplasmic membrane. Maintenance specificity was analyzed using a model precursor consisting mature part SecB-dependent maltose-binding protein (MalE) fused to signal peptide Tat-dependent TorA protein. The selectively specifically directed MalE into Tat pathway. characterization spontaneous mutant (TorA*), in which...

ABSTRACT The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these to Tat translocon. glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is periplasmic enzyme with NADP as It synthesized precursor an peptide shows all characteristics typical peptide....

Summary SecA is the precursor protein binding subunit of bacterial translocase, which consists SecY/E as integral membrane domain. an ATPase, and couples hydrolysis ATP to release bound proteins allow their proton‐motive‐force‐driven translocation across cytoplasmic membrane. A putative ATP‐binding motif can be predicted from amino acid sequence with homology consensus Walker A‐type motif. The role this domain not known. lysine residue at position 106 end glycine‐rich loop in Bacillus...

The SecA protein is a major component of the cellular machinery that mediates translocation proteins across Escherichia coli plasma membrane. secA gene from Bacillus subtilis was cloned and expressed in E. under control lac or trc promoter. temperature-sensitive growth secretion defects various mutants were complemented by B. protein, provided at moderate levels. Under overproduction conditions, no complementation observed. One main features ATPase activity which, together with protonmotive...