Purchase this article with an account.
M Traidej, AR Caballero, ME Marquart, BA Thibodeaux, RJ O'Callaghan; Molecular Cloning And Expression Analysis Of A Lysine Specific Proteinase, Protease IV From Pseudomonas aeruginosa . Invest. Ophthalmol. Vis. Sci. 2002;43(13):944.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:Protease IV has been identified as an extracellular lysine-specific protease of Pseudomonas aeruginosa. Computer analysis showed that the protease IV gene encoding a protein of 463 amino acids (48.2 kDa) contains a signal sequence, propeptide and mature protease domains. The only form of this protein that has been detected is the mature protease. In the present study, we investigate the maturation of protease IV using a new plasmid construct. Methods:The protease IV gene was cloned and expressed under control of a lac promoter in a protease IV-negative Pseudomonas species (P. putida) using an E. coli-Pseudomonas shuttle vector. The cloned protease IV gene product was analyzed in terms of its molecular properties, antigenicity and protease activity by multiple assays including N-terminal sequence analysis, substrate specificity, inhibitor reactivity and immunoblotting. Results:The expressed enzyme was fully processed into the active form. Expression of the protease IV gene in P. putida resulted in enzyme activity significantly greater (about 5-fold) than in P. aeruginosa. Western blot analysis also indicated a specific binding of polyclonal antibody directed against the mature protease of P. aeruginosa to the plasmid-encoded protease IV of P. putida. Analysis of the cell extract revealed proteins equivalent to the mature protease (26 kDa) and two major polypeptides representing the immature forms. One is the full-length protease IV (48 kDa) and the other probably contains the propeptide and mature protease domains (45 kDa). Conclusion:We demonstrate for the first time that protease IV is synthesized as a pre-proprotein that is processed and secreted out extracellularly as the mature protease. These results suggest that the processing of protease IV occurs intracellularly and possibly by autoprocessing.
This PDF is available to Subscribers Only