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) r esults in the release of periplasmic proteins into the culture medium. The BRP-mediated release of a periplasmic protein was investigated an d optimized. As a periplasmic model protein, the 50-kDa dimeric E. col i fimbrial molecular chaperone FaeE was used. Plasmids were constructe d for the simultaneous expression of the BRP and FaeE, controlled by i ndependently inducible promoters. The efficiency of FaeE release incre ased when the BRP was targeted by the unstable murein lipoprotein sign al peptide, instead of by its own stable signal peptide. Furthermore, optimal efficacy of FaeE release was found when cells of E. coli strai n C600 were used, which harboured one plasmid encoding both FaeE and B RP instead of two separate plasmids and which were cultured at 37 degr ees C in broth supplemented with MgCl2. Maximal production levels of 2 1 mg FaeE/1 culture were obtained.