OPTIMIZATION OF BACTERIOCIN RELEASE PROTEIN (BRP)-MEDIATED PROTEIN RELEASE BY ESCHERICHIA-COLI - RANDOM MUTAGENESIS OF THE PCLODF13-DERIVEDBRP GENE TO UNCOUPLE LETHALITY AND QUASI-LYSIS FROM PROTEIN RELEASE

Bacteriocin release proteins (BRPs) can be used for the release of het erologous proteins from the Escherichia coli periplasm into the cultur e medium, How-ever, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-IS sis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated pro tein release, the pCloDF13 BRP gene was subjected to random mutagenesi s by using PCR techniques, Mutated BRPs with a strongly reduced capaci ty to cause growth inhibition on broth agar plates were selected, anal yzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid;id cultures, A subset of the se BRP derivatives did not cause quasi-lysis and had only a small effe ct on growth but still functioned in the release of the periplasmic pr otein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture me dium, These BRP derivatives can be more efficiently used for extracell ular production of proteins by E. coli than can the original BRP.