Abstract:
β-lactams are an important class of antibiotics used in the treatment of various infections. These antibiotics are the most widely utilized by virtue of their high efficacy, low cost and safety profile. This widespread, heavy and sometimes irresponsible usage exerts considerable selection for resistance in bacteria. The most prevalent bacterial resistance mechanism to the β-lactam antibiotics is the production of β-lactamases. β-lactamases hydrolyze the amide bond of the four-membered β-lactam ring of the antibiotic rendering it inactive. Therefore, β-lactamase inhibitors emerge as an important research field to restore the therapeutic value of β-lactam antibiotics. Streptomyces clavuligerus produces a proteinaceous β-lactamase inhibitor (BLIP), which is a 165 amino acid protein. It inhibits several class A β-lactamases such as TEM-1 and SHV-1 β-lactamase with different affinities. In this study, in-vitro and in-vivo methods have been used to investigate β-lactamase inhibiton by BLIP. Periplasmic expression of BLIP has been achieved using two expression systems with different leader sequences, the native BLIP leader sequence and the pelB leader sequence. pUC18 vector was used for the expression of R-TEM-1 β-lactamase in E. coli BL21(DE3) cells. In-vivo binding experiments have been performed with cells expressing both BLIP and R-TEM-1 β-lactamase. Inhibition has been investigated by examining the growth characteristics of recombinant cells, colony forming units, SDS-PAGE analysis and measuring β-lactamase activity. In the presence of BLIP with the native leader sequence total β-lactamase activity dropped from 16450 U/L to 984 U/L. The reduction in β-lactamase activity was 40.9 per cent in the presence of BLIP with the native leader sequence while it was 62.4 per cent in the presence of BLIP with pelB leader sequence. Increasing the amount of BLIP to 531 μg and 325 μg, respectively resulted in 98 per cent d
