Optimization of bacteriocin-release-protein-induced protein release by Escherichia coli: extracellular production of the periplasmic molecular chaperone FaeE.
Expression of the pCloDF13-encoded bacteriocin-release protein (BRP) results in the release of periplasmic proteins into the culture medium. The BRP-mediated release of a periplasmic protein was investigated and optimized. As a periplasmic model protein, the 50-kDa dimeric E., coli fimbrial molecular chaperone FaeE was used. Plasmids were constructed for the simultaneous expression of the BRP and FaeE, controlled by independently inducible promoters. The efficiency of FaeE release increased when the BRP was targeted by the unstable murein lipoprotein signal peptide, instead of by its own stable signal, peptide. Furthermore, optimal efficacy of FaeE release was found when cells of E. coli strain C600 were used, which harboured one plasmid encoding both FaeE and BRP instead of two separate plasmids and which were cultured at 37 degrees C in broth supplemented with MgCl2. Maximal production levels of 21 mg FaeE/l culture were obtained.
van der Wal
F J
FJ
Department of Microbiology, Biocentrum Amsterdam, The Netherlands.
ten Hagen-Jongman
C M
CM
Oudega
B
B
Luirink
J
J
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D001692:Biological Transport; D003470:Culture Media / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010457:Peptidoglycan / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010457:Peptidoglycan; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C052368:cloacin DF13-encoded bacteriocin release protein, E coli; C070012:faeE protein, E coli; D007544:Isopropyl Thiogalactoside