TY - JOUR
T1 - Cloning and expression of calglandulin, a new EF-hand protein from the venom glands of Bothrops insularis snake in E. coli
AU - Junqueira-De-Azevedo, Inácio De L.M.
AU - Pertinhez, Thelma
AU - Spisni, Alberto
AU - Carreño, Flávia Regina
AU - Farah, Chuck S.
AU - Ho, Paulo Lee
N1 - Funding Information:
We thank Suely L. Gomes (IQ-USP) and Rita de Cássia Simão (IQ-USP) for providing calmodulin controls for biochemical assays. We would also like to thank Roy E. Larson (FMRP-USP) for some technical assistance and for the critical reading of the manuscript. This work was supported by grants from the Brazilian agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) and Fundação Butantan.
PY - 2003/5/30
Y1 - 2003/5/30
N2 - 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.
AB - 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.
KW - Bothrops insularis
KW - Ca binding protein
KW - Calglandulin
KW - EF-hand
KW - Expressed sequence tag
KW - Venom gland
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U2 - 10.1016/S1570-9639(03)00111-0
DO - 10.1016/S1570-9639(03)00111-0
M3 - Article
C2 - 12758151
AN - SCOPUS:0038305524
SN - 1570-9639
VL - 1648
SP - 90
EP - 98
JO - BBA - Protein Structure
JF - BBA - Protein Structure
IS - 1-2
ER -