Cloning and expression of calglandulin, a new EF-hand protein from the venom glands of Bothrops insularis snake in E. coli

Inácio De L M Junqueira-De-Azevedo, Thelma Pertinhez, Alberto Spisni, Flávia Regina Carreño, Chuck S. Farah, Paulo Lee Ho

Research output: Contribution to journalArticlepeer-review

Abstract

The EF-hand protein family is comprised of many proteins with conserved Ca2+-binding motifs with important biological roles in intracellular communication. During the generation of Expressed Sequence Tags (ESTs) from the venom glands of the Viperidae snake Bothrops insularis, we identified a cDNA coding for a putative Ca2+ binding protein with four EF-hand motifs, named here calglandulin. The deduced amino acid sequence displayed the greatest sequence similarity with calmodulin (59%), followed by troponin-C (52%). The encoded polypeptide was first expressed in Escherichia coli as a 6XHis-tagged fusion protein. The expressed protein was purified by Ni2+-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of α-helix as observed in calmodulin/calmodulin-like proteins. A polyclonal antiserum was generated in mice using this recombinant calglandulin. To investigate the tissue-specific biological occurrence of this protein, this antiserum was used in Western blot experiments, which revealed an immunoreactive band in samples of venom gland extracts from different snakes, but not in the crude venom or in brain, heart and other tissues. This exclusive occurrence suggests a specialized function of calglandulin in snake venom glands.

Original languageEnglish
Pages (from-to)90-98
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1648
Issue number1-2
DOIs
Publication statusPublished - May 30 2003

Keywords

  • Bothrops insularis
  • Ca binding protein
  • Calglandulin
  • EF-hand
  • Expressed sequence tag
  • Venom gland

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics

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