Cloning and expression of calglandulin, a new EF-hand protein from the venom glands of Bothrops insularis snake in E. coli.
The EF-hand protein family is comprised of many proteins with conserved Ca(2+)-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 Ca(2+) 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 Ni(2+)-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of alpha-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.
Junqueira-de-Azevedo
Inácio de L M
Ide L
Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil.
Pertinhez
Thelma
T
Spisni
Alberto
A
Carreño
Flávia Regina
FR
Farah
Chuck S
CS
Ho
Paulo Lee
PL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017837:Bothrops / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment
D003435:Crotalid Venoms; D011994:Recombinant Proteins; C475033:calglandulin, Bothrops insularis