Expression of functional scorpion neurotoxin Lqq-V in E.coli
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, CH19-B31, 1530-3rd Ave. South, Birmingham, AL 35294-2041, USA.Peptides (Impact Factor: 2.62). 02/2006; 27(1):49-54. DOI: 10.1016/j.peptides.2005.06.023
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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- "The interaction of scorpion toxins with their Na v receptor sites has been the focus of extensive research for several decades (reviewed in Rodriguez de la Vega and Possani 2007), yet molecular and structural details about these interactions were limited and have emerged only with the establishment of cloning procedures for the toxins and of an efficient bacterial expression system (LqhαIT anti-insect α-toxin, Gurevitz and Zilberberg 1994; Zilberberg et al. 1997; LqhIT2 and Lqh-dprIT 3 depressant β-toxins, Turkov et al. 1997; Strugatsky et al. 2005; Bj-xtrIT anti-insect excitatory toxin, Froy et al. 1999; Lqhβ1 and Css4 anti-mammalian β-toxins, Gordon et al. 2003; Cohen et al. 2005; Lqh3 α-like toxin, Karbat et al. 2007; Lqq5 and Aah2/Lqh2 anti-mammalian α-toxins, Banerjee et al. 2006; Kahn et al. 2009) and later in yeast (e.g., BmK M1 α-like toxin: Shao et al. 1999). From this point on the study of the toxins did not depend any longer on tedious purifications from crude venoms or chemical modifications that were limited to reactive residues (reviewed in Gurevitz 2012), but rather enabled substitution of any desired single or combination of amino acid residues and massive production of unmodified and mutant toxins for functional and structural analyses. "
ABSTRACT: Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Navs) at two pharmacologically distinct sites. Alpha toxins bind at receptor site 3 and inhibit channel inactivation, whereas beta toxins bind at receptor site 4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and competition for receptor sites at Nav subtypes. To elucidate the surface of interaction of the two toxin classes with Navs and clarify the molecular basis of varying toxin preferences, an efficient expression system was established. Mutagenesis accompanied by toxicity, binding, and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography, uncovered the bioactive surfaces of toxin representatives of all pharmacological groups. Exchange of external loops between channels that exhibit marked differences in sensitivity to various toxins accompanied by point mutagenesis highlighted channel determinants that play a role in toxin selectivity. These data were used in further mapping of the brain channel rNav1.2a receptor sites for the beta-toxin Css4 (from Centruroides suffusus suffusus) and the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). On the basis of channel mutations that affected Css4 activity, the known structure of the toxin and its bioactive surface, and using the structure of a potassium channel as template, a structural model of Css4 interaction with the gating module of domain II was constructed. This initial model was the first step in the identification of part of receptor site 4. In parallel, a swapping and a mutagenesis approach employing the rNav1.2a mammalian and DmNav1 insect Navs and the toxin Lqh2 as a probe were used to search for receptor site 3. The channel mapping along with toxin dissociation assays and double-mutant cycle analyses using toxin and channel mutants identified the gating module of domain IV as the site of interaction with the toxin core domain, thus describing the docking orientation of an alpha toxin at the channel surface.
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- "The E. coli AD949(DE3) strain is deficient in thioredoxin reductase and is therefore more suitable for the expression of peptides rich in disulfide structures. The recombinant peptide was obtained from the soluble fraction of the lysates and this finding is consistent with other two cysteine-rich peptides from scorpion venom described in the literature (Johnson et al., 2000; Banerjee et al., 2006), which are also obtained in soluble form when expressed in the same bacterial strain. The U 2 -SCRTX-Li1b recombinant peptide was the first ICK peptide from Loxosceles venom to be heterologously expressed. "
ABSTRACT: The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494(DE3) cells. The peptide was expressed by IPTG induction for 4 hours at 30°C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.
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- "The fusion protein was cleaved by thrombin, resulting in a yield of about 1.5 mg/L. The structure of the recombinat LqqV and its funtional activity was confirmed by NMR, CD spectroscopy and electrophysiology (Banerjee et al., 2006) One of the main limitations in the expression of spider toxins is the amount of active product. Although some reports have claimed a large production of spider toxins (Park et al., 2008), they have failed to prove the correct folding and activity of these arachnid peptides. "
ABSTRACT: This communication reviews most of the important findings related to venom components isolated from scorpions and spiders, mainly by means of gene cloning and expression. Rather than revising results obtained by classical biochemical studies that report structure and function of venom components, here the emphasis is placed on cloning and identification of genes present in the venomous glands of these arachnids. Aspects related to cDNA library construction, specific or random ESTs cloning, transcriptome analysis, high-throughput screening, heterologous expression and folding are briefly discussed, showing some numbers of species and components already identified, but also shortly mentioning limitations and perspectives of research for the future in this field.