Prokaryotic Recombinant Expression And Cellular Modulation Of Analgesic Scorpion Toxins

Ion channels are the electrical signals underlying cellular excitation. Scorpion toxins selectively interact on the membrane ion channels. In these toxins, many β-like anti-pain scorpion toxins targeted on the fourth site of voltage-gated sodium channels, and thus modulate activation, repolarization and inactivation of sodium channels. It may be its underlying analgesic mechanisms. In this paper, the prokaryotic recombinant expression and cellular modulation of three analgesic scorpion toxins was investigated.1.Construction of the recombinant expression for anti-scorpion toxinsAs far as we know there had a lot of methods of recombinant protein using Saccharomyces cerevisiae, Pichia and other eukaryotic expression of recombinant scorpion toxins, using insect, mammalian cells and so on, especially prokaryotic expression in E. coli. is easy to cultivate, the price is pretty low, contests expression has more advantages. So we built a scorpion toxin polypeptide expression system,using p ET-32 a as an expression vector, p ET system ever cloned in E.coli expression of recombinant protein expression system is relatively sound. Microbial culture of E. coli BL21(DE3) plus IPTG induction, duration and gradually narrow the optimal expression conditions, determine preliminary purified crude protein yield about 0.0885 g / 15 ml. Using SDS-PAGE electrophoresis, western-blot antibody experiment, AKTA protein purification, HPLC chromatography, whole-cell patch-clamp and other methods to study mechanisms of anti-pain toxins.(1) positive recombinant screening: the Bm K-AS, Bm K-AS1, Bm K-IT2 toxins gene fragments use Bam HⅠ, both within XhoⅠrestriction enzymes cut out sticky ends, with T4 DNA ligase connected the target gene with p ET-32 a vector, transformed into the TOP10 bacteria to preserve, and then extracted using SDS alkaline lysis plasmid DNA, recombinant plasmid vector molecules is more larger than empty plasmid, after running a gel electrophoresis showed significant recombinant plasmid hysteresis. Using SOB medium with ampicillin resistance successfully selected target plasmids of E. coli, the result is positive for expression of the toxin polypeptide.(2) IPTG induction of recombinant expression: isopropyl-β-D- thiogalactoside(IPTG) is similar to the natural β- galactosidase, which can not be broken down by enzymes and is a very effective inducer of Lac gene cluster, so is widely used in molecular biology and genetic engineering. Based on this we do that after one hour for microbial culture of E. coli plus IPTG induction, the sample bacteria expressing target proteins were broken,dissolved, centrifugated, and detected by electrophoresis. The results showed that the optimal recombinant expression of Bm K-AS was at 37 ℃ and 6 h, and Bm K-AS1 was at 30 ℃, and 6 h, and Bm K-IT2 was at 15 ℃ and 8 h.(3) Separation and purification of recombinant polypeptides: Nickel metal ion affinity column and HPLC were used for purification of recombinant toxins. The chromatographic results showed that nickel ion affinity interaction with the surface of the three recombinant toxins removal hybrid protein with 20 m M, 250 m M imidazole eluted target protein and collected. The best conditions for HPLC peak response of the three recombinant toxins using mobile was phase A and B ratio: 45%(1% aqueous trifluoroacetic acid) and 55%(60% acetonitrile).2. The anti-pain effects and cellular mechanisms of recombinant scorpion toxinsThe anti-pain effects and cellular mechanisms of three recombinant scorpion toxins were pursued by behavioral observation and patch-clamp electrophysiological recordings. Patch clamp results showed that the sodium currents of rat dorsal root ganglion(DRG) were inhibited significantly by recombinant anti-pain scorpion toxin Bm K IT2. The rat acetate-induced pain was inhibited by intraperitoneal injection of 1 mg / ml(200(30)l) of three recombinant scorpion toxins in various degrees. These differences may be related to sequence, structure and its interaction with the sodium channels of scorpion toxins.In summary, the scorpion toxin is one kind of neuropharmacology molecular tools for ion channels. In this paper, by constructing a prokaryotic cell exogenous expression system, and optimizing the three scorpion toxin polypeptide soluble expression conditions, proved the differences between the anti-pain activity of three peptides scorpion toxins. The results help to advance the state of the natural scorpion toxin expressed in low abundance, no other active chemical synthesis, structural analysis and transformation of technical problems, for further study the pain associated membrane ion channel structure and function provides a scientific and technical basis.