Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli.
The extracellular zinc-metalloprotease of Vibrio 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 two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.
Zhang
Fengli
F
UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
Chen
Jixiang
J
Chi
Zhenming
Z
Wu
Long-Fei
LF
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D045726:Metalloproteases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics; D015032:Zinc / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C500552:EmpA protein, Vibrio anguillarum; D045726:Metalloproteases; D015032:Zinc