cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus
Abstract
AIM:
To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production.
METHODS:
PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template. The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells.
RESULTS:
A novel cDNA sequence encoding akitonin beta was found and accepted by GenBank (accession number AF387100). Akitonin beta consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin beta expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro.
CONCLUSION:
A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.
Keywords:
To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production.
METHODS:
PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template. The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells.
RESULTS:
A novel cDNA sequence encoding akitonin beta was found and accepted by GenBank (accession number AF387100). Akitonin beta consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin beta expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro.
CONCLUSION:
A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.