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Cloning of serine protease cDNAs from Crotalus durissus terrificus venom gland and expression of a functional Gyroxin homologue in COS-7 cells.
- Source :
-
Toxicon : official journal of the International Society on Toxinology [Toxicon] 2009 Aug; Vol. 54 (2), pp. 110-20. Date of Electronic Publication: 2009 Mar 31. - Publication Year :
- 2009
-
Abstract
- Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.
- Subjects :
- Amino Acid Sequence
Animals
Blotting, Western
COS Cells
Chlorocebus aethiops
Cloning, Molecular
Crotalid Venoms enzymology
Crotalid Venoms genetics
DNA, Complementary genetics
Electrophoresis, Polyacrylamide Gel
Escherichia coli metabolism
Esterases chemistry
Esterases metabolism
Exocrine Glands enzymology
Gene Library
Genetic Vectors
Mice
Molecular Weight
Plasmids genetics
Recombinant Proteins genetics
Serine Endopeptidases genetics
Crotalid Venoms biosynthesis
DNA, Complementary biosynthesis
Exocrine Glands chemistry
Serine Endopeptidases biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1879-3150
- Volume :
- 54
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Toxicon : official journal of the International Society on Toxinology
- Publication Type :
- Academic Journal
- Accession number :
- 19341755
- Full Text :
- https://doi.org/10.1016/j.toxicon.2009.03.022