Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis

 

Herlinda Clement¹, Vianey Flores¹, Guillermo De la Rosa¹, Fernando Zamudio¹, Alejandro Alagon¹, Gerardo Corzo¹

 

1 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Av. Universidad 2001, Cuernavaca 62210, Morelos, Mexico

 

ABSTRACT

 

Background

The 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.

 

Method

Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC).

 

Results

The 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.

 

Conclusion

HisrMlat1 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.

 

Key words: Micrurus laticorallis; Protein folding; Recombinant; Elapid; Toxin; Protein recognition

 

Funding

This work was financed by grants from Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) number IN204415 and from SEP-CONACyT number 240616 to GC.

 

Ethics approval and consent to participate

The present study was approved (project #271) by the Ethics Committee of Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Committee of Animal Welfare, keeping the number of animals to a minimum required to validate the experiments.

 

Received: April 5, 2016.

Revised: September 2, 2016.

Accepted: September 9, 2016.

 

Correspondence: corzo@ibt.unam.mx

 

Authors’ contributions

HC constructed the gene within the vector and expressed the proteins. VF purified the recombinant neurotoxins. GR performed the LD₅₀ experiments. FZ performed the mass spectrometric analysis. AA contributed to analyzing the results and to writing the manuscript, GC conceived and coordinated the study, and contributed to designing the experiments and to writing the final version of the manuscript. All authors reviewed the results and approved the final version of the manuscript.

 

Competing interests

The authors declare that they have no competing interests.

 

doi: 10.1186/s40409-016-0080-9