Investigation of the in vivo interaction between β-lactamaseand its inhibitor protein

The affinity of ß-lactamase inhibitory protein (BLIP) for TEM-1 ß-lactamase has raised hopes in the challenge of protein-based inhibitor discovery for ß-lactamase-mediated antibiotic resistance. Currently, the effect of the formation of the ß-lactamase:BLIP complex in vivo in ß-lactam resistant bacteria is an open question. The scarcity of information to the extent to which BLIP can impair ß-lactamase activity inside cells has urged us to assess the in vivo efficacy of BLIP as a potent ß-lactamase inhibitor. To this end, ß-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of ß-lactamase and BLIP and the formation of the TEM-1 ß-lactamase:BLIP complex in the periplasmic space of E. coli were verified by electrophoretic and Western blot techniques. Growth profiles of the cells expressing both ß-lactamase and its protein inhibitor, complemented with ß-lactamase activity measurements, suggested that BLIP synthesis retarded cell growth and reduced ß-lactamase activity. Although co-expression of ß-lactamase and its protein inhibitor did not completely impair cell growth, the specificity of BLIP enabled it to bind ß-lactamase in the bacterial periplasm, regardless of the crowding components.