Expression, purification and virucidal activity of two recombinantisoforms of phospholipase A2from Crotalus durissus terrificus venom

Abstract

AbstractThe global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have becomea public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccinesagainst arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds.Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component ofCrotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide(crotapotin) and a phospholipase A2(PLA2-CB). We showed previously the antiviral effect of PLA2-CB against denguevirus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in aprokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimizedand cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressedin the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturingconditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatographymatrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, whichconsisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combinationwith the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer.The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, denguevirus, yellow fever virus and Zika virus