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

Abstract

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