Expression, Processing And Export Of Vibrio Anguillarum Metalloprotease In Escherichia Coli, Purification And Characterization Of Recombinant Metalloprotease

The maricultural industry has been developing very rapidly in last two decades in China and sea-food yields in China have become the biggest in the world. However, the industry is frequently plaqued by bacterial infections, particularly vibriosis mainly caused by Vibrio anguillarum, leading to extensive economical losses. So far, much less has been known about the virulence factors that are involved in the pathogenesis of marine Vibrio spp. Studies on pathogenicity of various marine Vibrio spp showed that their extracellular enzymes including protease, haemolysin, phospholipase and chitinase are ones of the virulence factors. The zinc metalloprotease has been identified as one of the important virulence factors secreted by V. anguillarum.The extracellular zinc-metalloprotease of V. anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed.To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in...