Expression, Purification And Characterization Of The Scorpion Insect Toxin BmK IT From Buthus Martensii Karsch

Scorpion is an animal which preys on mollusk insect. A series of toxin were brought in its body for survival and defense among long term evolution. Mainly neurotoxic proteins or peptides can interact specifically with various ionic channels in excitable membranes, so they have been used as valuable probes for the elucidation of structure and function of ion channels. At the same time, scorpion neurotoxins are very useful models for studying protein structure-function relationship. Especially, all kinds of insect-selective scorpion neurotoxins display no or less toxicity toward mammals but are specifically active toward insects. Therefore, they are widely used for studying biological pesticides and anti-insect transgenic plants. Therefore, more and more biologists focus on scorpion insect neurotoxin.An excitatory insect toxin gene, BmK IT, from Buthus martrnsii Karsch which is a sort of scorpion mainly distributed in our country was cloned previously in our lab. This toxin is composed of 69 amino acid residues and is cross-linked by four disulfide bridges. In this study, BmK IT was soluble expressed in procaryotic cell, and was producted by a series of purified approach, for example, optimization of buffer, intein-self cleavage and protein oxidative refolding. Meanwhile, the pilot study on catalysis of E. coli disulfide isomerase DsbC for folding of iBmK IT was performed. Further more, analysis of anti-insect bioassay, whole cell patch-clamp recording, circular dichroism spectrum were performed to determine the biochemical characterization of it.First, the recombinant expression plasmid pTWIN1-BmK IT-His₆ was constructed and the fusion protein DnaB- BmK IT-His₆ was expressed by it. A His₆ tag was fused to the C terminus of DnaB- BmK IT. rBmK IT was eluted after intein self-cleavage on the Ni-NTA resin. However, it was found that most of rBmK IT was existed as polymer and a little monomer by size-exclusion chromatography. The results of anti-insect bioassay of monomer rBmK IT indicated that the LT₅₀ of 1.0 and 2.0 nmol/g rBmK IT for cotton worm were 67±1hå’Œ47±1 h, respectively.Second, the folding behavior of rBmK IT during the purification was analyzed farther. The DnaB moieties in the DnaB- rBmK IT fusion are biologically active since they can self-cleave efficiently at pH 6.5. By contrast, the released proteins rBmK IT was precipitated rapidly in the same experiment. Further more, assay of free sulfhedryl groups indicated that the target protein expressed in E. coli cell was unfolded, and that some disulfide bonds were formed during IMAC (Immobilized Metal ion Affinity Chromatography). Thus, the soluble fusion protein was named as "soluble inclusion bodies", which included of folded carrier protein DnaB and unfolded/misfolded target protein rBmK IT. Consequently, the recombinant protein oxidative refolding was performed in the optimized condition (50 mmol/L Tris-HCl, pH 8.5, 2.0 mmol/L GSH, 0.5 mmol/L GSSG). An excited result appeared, in which rBmK IT was efficiently refolded and existed as monomer. Another important quality of the resulting rIT-His₆ is its high level of thermostability since it could tolerate temperatures up to 100℃. Consequently, a simple and convenient approach was used to purify rIT-His₆. The crude extract was first applied to IMAC. After washing, both oxidative refolding of interest protein and subsequent intein self-cleavage were performed on a packed Ni-NTA resin column. And then the solution eluted was treated by incubation at 80℃for 20 min. Finally, purified rBmK IT was obtained by centrifugation. The final product - His₆ tagged rBmK IT by this procedure was about 1.5 mg from 250 ml cultured cells.At the same time, the E.coli disulfide isomerase DsbC was cloned, soluble and functional expressed and purified. And catalysis of E.coli disulfide isomerase DsbC for folding of rBmK IT was performed. The result showed that rDsbC could enhance the refolding of rBmK IT in vitro and the reaction condition should be further optimized. However, expected result was not gotten from the experiment of co-expression of rBmK IT with DsbC in vivo. Because rBmK IT was co-expressed with DsbC in the cytoplasm, which is a reductive condition and is disadvantageous for formation of disulfide. So the future work is that rBmK. IT should be co-expressed with DsbC in the periplasm by secretive expression vector.The separation and culture of nerve cells from cotton worm (Helicoverpa armigera) were performed and this work paved the way for electrophysiological studies of rBmK IT by whole cell patch-clamp technique.Finally, Crystallization of rBmK IT was performed using the hanging-drop method. 46 conditions (Crystal Screening Kit, Hampton Research) were used in order to get high quality, well repeated crystals. An awl-formed crystal of it could be obtained in the buffer 0.1 mol/L Tris-HCl, pH 8.5, containing 0.2 mol/L tri-Sodium Citrate dihydrate, 30% v/v Polyethylene Glycol 400 at room temperature and the protein was dissolved in ddH₂O.