Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii

AbstractEntomopathogenic bacteria of the genusPhotorhabdus secrete protease  S (PrtS), which is considered a virulence factor. We found that in thePhotorhabdus genomes, immediately after theprtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor fromPhotorhabdus laumondii subsp.laumondii TT01 was cloned and expressed inEscherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0  ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity ofP.  laumondii culture fluid (half-maximal inhibition concentration 3.9  ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting thatP.  laumondii cells produce Phin. Thus, theprtS genes in entomopathogenic bacteria of the genusPhotorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of prot...
Source: Biochemistry (Moscow) - Category: Biochemistry Source Type: research