cDNA sequencing of fragments from fibrinolytic gene (F-4) from Cerastes cerastes venom gland

Abstract

A fibrinolytic anticoagulant (F-4) was previously isolated by Daoud et al; (1986) from Cerastes cerastes venom. In this study, isolation and sequencing of gene fragments of this F-4 protein was attempted. Primers utilized in the polymerase chain reactions were obtained from two sources: P1, a primer that was previously used in isolating other fibrin(ogen)olytic metalloprotease. The other primers; P2, P3 and P4 were designed using doprimer program. RT-PCR using P1 and P2 primers gave a band of 350 bp. P4 and P3 primers gave a band of 60 bp. P1 and P3 primers gave a band of 400 bp. Sequence analysis and alignment using bioinformatic programs indicated that samples 1, 2 and 3 bear significant homology to the metalloprotease family of snake venom sequences deposited in the Genbank. Translation to the amino acid sequence and alignment using protein database showed strong homology with fibrinolytic metalloproteases. Conservative domain database analysis indicated that the three sequenced samples belong to reprolysin family, which is a snake venom endopeptidase requiring zinc for catalysis. Predict protein algorithm indicated one cysteine disulfide bond in sample 3. Sample 1 was described as mixed protein with 22% helix 35% extended and 41% loop structure. Sample 2 was described as all-alpha protein with 76% helix and 24% loop. Sample 3 was described as alpha–beta protein with 30% helix, 27% extended and 41% loop. The sequenced fragments of the protein were generally described as globular and seemed to be truncated of one gene related to a fibrinolytic protein.

Keywords: Fibrin(ogen) lysis, zinc metalloprotease, primer design, cDNA sequencing and conserved domain

Egyptian Journal of Biochemistry and Molecular Biology Vol. 24 (2) 2006: pp. 154-177