cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus.
OBJECTIVE
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.


CONCLUSIONS
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.
Zha
Xiang-dong
XD
School of Life Science, University of Science and Technology, Hefei 230026, China. xdcha@163.com
Liu
Jing
J
Xu
Kang-sen
KS
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003435:Crotalid Venoms / Q000737:chemistry*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D037181:Lectins, C-Type / Q000737:chemistry*; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D017421:Sequence Analysis
D003435:Crotalid Venoms; D018076:DNA, Complementary; D037181:Lectins, C-Type; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins