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

Abstract

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