Effect of simultaneous beta-lactamase inhibitor protein expression on cellular beta-lactamase activity

Beta-lactam antibiotics are commonly used in the treatment of bacterial infections. However, due to underuse and misuse of beta-lactam antibiotics, bacteria have developed mechanisms that counteract beta-lactam antibiotics. The most common mechanism is production of TEM-1 beta-1actamase that hydrolyzes the beta-lactam ring and enables bacteria to gain antibiotic resistantance. Beta-lactamase-inhibitory protein (BLIP), a 165 amino acid protein produced by Streptomyces clavuligerus, is a high affinity beta-lactamase inhibitor. In this study, the inhibitory effect of BLIP was investigated by examining growth profiles, cell viability and in-vivo beta-lactamase activity of recombinant cells simultaneously expressing beta-lactamase and BLIP. beta-lactamase activity per viable cell was determined in the absence and presence of BLIP. The presence of both BLIP and beta-lactamase was verified by SDS-PAGE and Native-PAGE analysis. In the absence of BLIP, E. coli BL21(DE3) (pUC18 + pET-26EA) and E. coli BL21(DE3) (pUC18 + pET-26b(+)) cells showed comparable beta-lactamase activity. The activity of beta-lactamase in the periplasmic extract of E. coli BL21(DE3) (pUC18 + pET-26EA) cells decreased considerably when R-TEM-1 beta-lactamase and BLIP were expressed simultaneously.