Molecular cloning, expression and characterization of three short chain α-neurotoxins from the venom of sea snake -: Hydrophiinae Hydrophis cyanocinctus Daudin

被引:5
作者
Peng, LS [1 ]
Zhong, XF [1 ]
Huang, YS [1 ]
Zhang, Y [1 ]
Zheng, SL [1 ]
Wei, JW [1 ]
Wu, WY [1 ]
Xu, AL [1 ]
机构
[1] Sun Yat Sen Univ, Coll Life Sci, Dept Biochem, Open Lab Marine Funct Gen State High Tech Dev, Guangzhou 510275, Peoples R China
基金
中国国家自然科学基金;
关键词
Hydrophiinae Hydrophis cyanocinctus Daudin; short chain alpha-neurotoxins; fusion expression; LD50; blockade; neuromuscular transmission;
D O I
10.1016/j.toxicon.2003.10.002
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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 Lys(46) and Ser(46), respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp(31) and Arg(33). 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. (C) 2003 Elsevier Ltd. All rights reserved.
引用
收藏
页码:753 / 761
页数:9
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