Recombinant Ophiophagus hannah Long neurotoxin OH-5
Product Specs
Target Background
Q&A
What structural features differentiate OH-5 from other α-neurotoxins in snake venoms?
OH-5 is a 72-residue polypeptide with 10 conserved cysteines forming five disulfide bonds, characteristic of long-chain α-neurotoxins (LNTXs) . Its tertiary structure features three β-stranded loops (loops I-III), with loop II containing critical cationic residues (Arg-35, Arg-37) for nicotinic acetylcholine receptor (nAChR) binding . Unlike short-chain neurotoxins, OH-5 has an additional C-terminal tail (residues 61–72) that enhances receptor affinity . Sequence alignment reveals 89% homology with Oh-4 (a paralog in O. hannah venom) but only 60% similarity to toxins a/b from the same species, suggesting functional divergence .
Methodological Insight: Use Edman degradation coupled with trypsin/V8 protease digestion for sequencing . Validate disulfide connectivity via tandem mass spectrometry under non-reducing conditions .
How is recombinant OH-5 expressed and purified for functional studies?
rOH-5 is typically produced in E. coli BL21(DE3) using pET vectors. Codon optimization is critical due to the high cysteine content (13.9%) . After IPTG induction, soluble rOH-5 is extracted via Ni-NTA affinity chromatography (His-tag) and further purified by reversed-phase HPLC (C18 column, 0–60% acetonitrile gradient) . Refolding is achieved using a glutathione redox system to ensure proper disulfide bonding .
Key Data:
| Parameter | Value | Source |
|---|---|---|
| Expression Yield | 8–12 mg/L culture | |
| Purity Post-HPLC | >95% (SDS-PAGE) | |
| LD50 (mice, i.m.) | 0.12 µg/g |
What assays confirm rOH-5’s bioactivity and specificity?
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Electrophysiology: Patch-clamp recordings of mouse α12βγδ nAChRs in HEK293 cells show rOH-5 inhibits ACh-induced currents (IC50 = 3.2 nM) .
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Lethality Testing: Intraperitoneal injection in mice (1.5 LD50 = 0.18 µg/g) with survival monitored for 48 h .
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Receptor Binding: Radiolabeled rOH-5 competes with α-bungarotoxin for Torpedo nAChR sites (Kd = 1.8 nM) .
Troubleshooting: False negatives in lethality assays often arise from incomplete refolding. Validate with circular dichroism (CD) spectroscopy to ensure β-sheet content matches native toxin .
How to resolve contradictions in reported receptor binding affinities for rOH-5?
Discrepancies in IC50 values (e.g., 3.2 nM vs. 8.7 nM ) stem from:
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Receptor Subtype: α12βγδ (muscle) vs. α7 (neuronal) nAChRs.
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Assay Conditions: 22°C vs. 37°C, altering toxin-receptor kinetics.
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Post-Translational Modifications: Non-native disulfide bonds in E. coli-derived rOH-5.
Resolution Strategy:
What experimental designs optimize rOH-5’s cytolytic activity studies?
While OH-5 primarily targets nAChRs, recombinant variants exhibit cardiotoxin-like cytolysis (e.g., 40% hemolysis at 10 µM ). To isolate cytolytic effects:
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Membrane Depolarization Assays: Use DiSC3(5) dye in erythrocytes to quantify membrane disruption .
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Site-Directed Mutagenesis: Replace hydrophobic residues in loop I (Phe-11, Trp-18) to dissect neurotoxic vs. cytolytic domains .
Critical Controls:
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Include α-bungarotoxin (non-cytotoxic) to confirm assay specificity.
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Test temperature dependence (4°C vs. 37°C) to distinguish pore formation from enzymatic activity .
How does rOH-5 inform antivenom development against king cobra envenoming?
rOH-5’s epitopes guide cross-reactive antivenom engineering. Naja kaouthia antivenin binds OH-5 via conformational epitopes in loops II/III, achieving 50% lethality neutralization at 1:10 (w/w) . Humanized scFvs (e.g., NkLN-HuScFv) targeting Arg-35/37 increase survival to 85% in murine models .
Table: Antivenom Neutralization Efficacy
| Antivenom | ED50 (mg venom/mg IgG) | Survival (1.5 LD50) |
|---|---|---|
| N. kaouthia F(ab’)2 | 0.45 | 50% |
| NkLN-HuScFv | 0.12 | 85% |
Mechanistic Insight: Molecular docking shows NkLN-HuScFv’s CDR3 region forms salt bridges with OH-5’s Arg-35 (ΔG = −9.8 kcal/mol) .
