Expression of functional scorpion neurotoxin Lqq-V in E.coli.
ABSTRACT We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 degrees C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the alpha-toxin activity of the purified recombinant protein.
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ABSTRACT: The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.Biochimica et Biophysica Acta 09/2007; 1770(8):1161-8. DOI:10.1016/j.bbagen.2007.04.006 · 4.66 Impact Factor
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ABSTRACT: BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b5 fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a “misfolded” form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b5 fusion protein system for the efficient production of peptides for high resolution NMR studies.Protein Expression and Purification 04/2010; 70(2-70):137-142. DOI:10.1016/j.pep.2009.10.017 · 1.51 Impact Factor
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ABSTRACT: Heteroscorpine-1 (HS-1) was identified as a member of the scorpine family. HS-1 shows insecticidal activities, exhibiting a low median lethal dose (LD50) in mealworm (Tenebrio molitor L.) and inhibitory activities against Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In this study, a recombinant HS-1 (rHS-1) was produced by overexpression in E. coli. A large yield of product was obtained. The structure of purified rHS-1 was confirmed through mass spectrometry. Both anti-crude venom and anti-rHS-1 antibodies specifically recognized rHS-1, suggesting its structural similarity. Reactivated rHS-1 caused roughening and blebbing of bacterial cell surfaces. It showed higher activity than that of pre-refolded protein. Antisera raised against a partially purified and mis- or unfolded peptide can inhibit relevant bioactivity.Biochemical Genetics 07/2014; 52(11-12). DOI:10.1007/s10528-014-9660-y · 0.82 Impact Factor