Genomic DNA Sequences And Expression, Purification Of BmαTX14 Neurotoxin From Scorpion Buthus Martensii Karsch

The Asian scorpion Buthus martensi Karsch (BmK) is widely distributed in China, the venom is rich sources of toxic polypeptides that affect ion-channel function of excitable cells, containing 20-80 amino acid residues cross-linked by four or three disulfide bridges. Four different families of toxins have been described, which specifically interact with ion channels: Na~+, K~+, Cl~-, Ca~2+ and are proved to be important tools for clarifying the physical effects and functional mapping of ion channels. The Asian scorpion BmK is not dangerously venomous for mammal; in fact, its components have demonstrated anti-hyperalgesic and anti-epileptic effect. However, the toxic peptides are too complex to purification and difficult to obtain enough polypeptides for the study.According to the cDNA sequences of bmatx14 which was isolated from the cDNA library of Chinese scorpion BmK before, the primers were designed and full-length sequence of bmatx14 was amplified from the scorpion genome. The genomic amplification of bmatx14 by PCR yielded a predominant band of 855 bp. Sequence analysis of this fragment confirmed that the genomic gene of bmatx14 consisted of two exons disrupted by an intron of 408 bp, similar with other a-scorpion toxin genomes. This intron is in the sequence encoding the signal peptide, after thefirst base (G) of a Gly16 codon, beginning with GT and ends with AG. The intron has the sequences of the 5-' splice donor: 5'-AG I gtaaga-3' and that of the 3-' splice acceptor: 5'-tgacag | GA-3' and that of branch point: 5'-taat-3'. The genomic organization of bmatx14 proved to be a membership belonging to a-type sodium ion-channel toxin.Scorpion toxins are the main useful component of whole scorpion. However, it is difficult to purify for the complexity of toxin component. Meanwhile, the content of useful toxins is limited. All of these limit the use of scorpion toxins as medicine. Therefore, our research builds up an efficient expression and purification system based on the use of Pichia yeast expression system. The cDNA encoding mature peptide of BmaTX14 was amplified by PCR and inserted into the pPIC9K expression vector, while the His-tag was designed to be inserting between the signal peptide of the vector and the mature peptide. Then the recombination vector was transformed to Pichia pastoris GS115 strains. After several screenings of grads of G418, the transformed strains containing multiple inserts were chosen to be induced with methanol in shake flask culture. The secreted rBmaTX14 protein was expressed in the medium and most impurities were eliminated by using Ni-NTA-agarose, polyethylene glycol precipitation and gel filtration chromatography. The concentration of rBmaTX14 purified was determined by the method of Bradford, then by the calculation of the proportion of purification and condense, the expression quantity of rBmaTX14 in the medium was nearly 120mg/L. Meanwhi le, the purified rBmaTX14 proved to have the anti-insect activity by toxicity assay and the FPU was 3.885 g/100mg. However, rBmaTX14 did not exhibit any toxicity to mammals.It is the main method that using E.coli expression system to gain recombined scorpion toxins today. We utilized the P .pastoris expression system to express the active sodium ion-channel toxin, BmaTX14, efficiently, which pioneered the wide prospect of the exploitation and utilization of the abundant scorpion medicaments.