We have previously reported (SAB meeting, 2008) the first cloning of isoenzymes of phospholipase A2 (PLA2) from Bothrops diporus (Yarará chica), a common viper of Argentina, and the expression of one of these in inclusion bodies fused to a N-terminal 6xhistidine-tag and a thrombin recognition sequence. Hydrolysis of dLPC monolayers upon addition of that construction was not observed. This, along with some reports that indicate the importance of a native N-terminal sequence to obtain full interfacial catalysis1, prompted us to set up a system to obtain native sequence protein.
Expression of one of these isoenzymes fused to an ubiquitin moiety was successfully achieved. Fusion protein was found in inclusion bodies. Its reduction and complete denaturation were carried out to solubilize the Ubiquitin-PLA2 protein. After this, solution was dialyzed against oxidation buffer, followed by a ten-fold dilution. To cleave off Ubiquitin and obtain PLA2 with native N-terminus, peptidase USP2cc2 was added. After this, PLA2 activity was detected as hydrolysis of monolayers of dLPC (substrate), followed by the surface barostat technique and the optimal dLPC lateral pressure for hydrolysis was determined3. No PLA2 activity could be detected prior the addition of USP2cc.
To our knowledge this is the first report of heterologous production of an active phospholipase A2 from Bothrops diporus and utilization of the Ubiquitin/USP2cc system to obtain any PLA2 in E. coli.
