Archives of Virology

, Volume 164, Issue 4, pp 1159–1171 | Cite as

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom

  • Raquel Rinaldi Russo
  • Nilton Nascimento dos Santos Júnior
  • Adélia Cristina Oliveira Cintra
  • Luiz Tadeu Moraes Figueiredo
  • Suely Vilela Sampaio
  • Victor Hugo AquinoEmail author
Original Article


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 A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-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.



Dengue virus


Zika virus


Yellow fever virus


Chikungunya virus


Phospholipase A2


Secreted phospholipase A2


Snake venom phospholipase A2




Phospholipase A2 crotoxin B


Phospholipase A2 crotoxin B isoform 1


Phospholipase A2 crotoxin B isoform 2


Recombinant phospholipase A2 crotoxin B isoform 1


Phospholipase A2 crotoxin B isoform 2


Stereo-specific number 2


Polyhistidine tag


Luria-Bertani medium


Tris-buffered saline


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide


Dimethyl sulfoxide


50% cytotoxic concentration


Plaque-forming units


Isopropyl β-D-1-thiogalactopyranoside


5-Bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium


Phenylmethylsulfonyl fluoride




Sodium dodecyl sulfate


Polyacrylamide gel electrophoresis


Immunoglobulin G


Standard deviation


3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate



This work was supported by the Sao Paulo Research Foundation (FAPESP), grant no. 2014/02438-6) and the National Council of Technological and Scientific Development (CNPq) (grant no. 479512/2012-4). RR was supported by a FAPESP scholarship (grant no. 2012/12605-1) and VHA holds a CNPq-PQ fellowship (grant no. 306471/2017-5).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal experiments

All animal experiments were performed according to the guidelines of the Brazilian College of Animal Experimentation and were approved by the Ethical Committee on Animal Experimentation of the Campus of Ribeirao Preto, University of Sao Paulo (CEUA/USP-RP, Permit. Nº 12.1.1854.53.8).

Supplementary material

705_2019_4172_MOESM1_ESM.pdf (172 kb)
Supplementary material 1 (PDF 171 kb)
705_2019_4172_MOESM2_ESM.pdf (476 kb)
Supplementary material 2 (PDF 475 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Raquel Rinaldi Russo
    • 1
  • Nilton Nascimento dos Santos Júnior
    • 2
  • Adélia Cristina Oliveira Cintra
    • 3
  • Luiz Tadeu Moraes Figueiredo
    • 4
  • Suely Vilela Sampaio
    • 3
  • Victor Hugo Aquino
    • 1
    Email author
  1. 1.Laboratory of Virology, Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirao PretoUniversity of Sao PauloRibeirao PretoBrazil
  2. 2.Laboratory of Neuro-immuno-endocrinology, Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical SchoolUniversity of São PauloRibeirao PretoBrazil
  3. 3.Laboratory of Toxinology, Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirao PretoUniversity of Sao PauloRibeirao PretoBrazil
  4. 4.Virology Research Center, Ribeirao Preto Medical SchoolUniversity of Sao PauloRibeirao PretoBrazil

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