Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A(2) from Crotalus durissus terrificus venom

Autor(es)

Russo, Raquel Rinaldi ^[1] ; dos Santos Junior, Nilton Nascimento ^[2] ; Oliveira Cintra, Adelia Cristina ^[3] ; Moraes Figueiredo, Luiz Tadeu ^[4] ; Sampaio, Suely Vilela ^[3] ; Aquino, Victor Hugo ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Clin Anal Toxicol & Food Sci, Lab Virol, Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Neurosci & Behav Sci, Lab Neuroimmunoendocrinol, Ribeirao Preto, SP - Brazil

^[3] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Clin Anal Toxicol & Food Sci, Lab Toxinol, Ribeirao Preto, SP - Brazil

^[4] Univ Sao Paulo, Ribeirao Preto Med Sch, Virol Res Ctr, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: ARCHIVES OF VIROLOGY; v. 164, n. 4, p. 1159-1171, APR 2019.
• Citações Web of Science: 2

Resumo

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against 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 of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A(2) (PLA(2)-CB). We showed previously the antiviral effect of PLA(2)-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA(2)-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA(2)-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with 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, dengue virus, yellow fever virus and Zika virus. (AU)

• Processo FAPESP: 14/02438-6 - Estudos com Bunyaviridae causadores de doença
• Beneficiário: Luiz Tadeu Moraes Figueiredo
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 12/12605-1 - Clonagem e expressão da fosfolipase A2-CB do veneno da serpente Caudisona durissa terrificus e avaliação da sua atividade antidengue e antifebre amarela
• Beneficiário: Raquel Rinaldi Russo
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto