Molecular cloning, expression and characterization of three short chain alpha-neurotoxins from the venom of sea snake--Hydrophiinae Hydrophis cyanocinctus Daudin.
Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain alpha-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys46 and Ser46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp31 and Arg33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified alpha-neurotoxins, and no significant structural differences in these recombinant proteins are observed.
Peng
Li-Sheng
LS
The Open Laboratory for Marine Functional Genomics of State High-Tech Development, Department of Biochemistry, College of Life Science, Sun Yat-Sen University, Guangzhou 510275, People's Republic of China.
Zhong
Xiao-Fen
XF
Huang
Yu-Shan
YS
Zhang
Yuan
Y
Zheng
Sui-Lan
SL
Wei
Jian-Wen
JW
Wu
Wen-Yan
WY
Xu
An-Long
AL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D004546:Elapid Venoms / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D017815:Elapidae; D005260:Female; D015723:Gene Library; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000187:drug effects; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D010791:Phrenic Nerve / Q000187:drug effects; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D004546:Elapid Venoms; D009498:Neurotoxins