Abstract
BackgroundThe cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production.MethodCysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC).ResultsThe cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa.ConclusionHisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.
Highlights
The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; their low concentration in the venom may hamper the production of efficient elapid antivenoms
After the gene was amplified, the product with the expected size (~230 bp) was purified via the High Pure Plasmid Isolation Kit (Roche, Germany) cloned into PCR®2.1-TOPO vector (Invitrogen, USA) and its sequence was verified and the plasmid pQ30XaMlat1 was constructed (Fig. 1c). This plasmid was confirmed to contain the DNA sequence to code a segment of six histidines followed by four amino acids corresponding to cleavage site of the enzyme Factor Xa, and the 60 amino acid residues corresponding to the mature toxin isolated from M. laticorallis (Fig. 1d)
As mentioned in the introductory section, it is an important contribution due to the fact that the literature has not reported in vivo toxicity of recombinant coral snake neurotoxins possibly because of wrongly folded toxins
Summary
The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; their low concentration in the venom may hamper the production of efficient elapid antivenoms. Snake neurotoxins that affect acetylcholine receptor (AChR) have served as a powerful tool to elucidate, both pharmacologically and functionally, different subtypes of AChR Due to their high toxicity, these curare-mimetic toxins represent an important target for the antivenom industry [1, 2]. In North America, Micrurus laticollaris is endemic in Mexico, and its principal habitat is the tropical deciduous forest along the Balsas River in south-central Mexico, which flows through the Mexican states of Puebla, Morelos, Guerrero, and Michoacan, and empties into the Pacific Ocean [3,4,5] The venom of this elapid causes neuromuscular blockade in mammalians, which is preceded by a presynaptic effect that facilitates acetylcholine neurotransmitter release [6]. We report a heterologous expression system for obtaining recombinant HisrMlat or rMlat with structural and functional characteristics similar to the native one, as well as the antibody recognition proving that the recombinant HisrMlat can be used as an immunizing agent
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