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

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.

Horse immunization with short-chain consensus α-neurotoxin generates antibodies against broad spectrum of elapid venomous species

“…Stored pQE30/ScNtx 10 , pQE30/MlatA1 49 , and pQE30/r.D.H 50 plasmids were transformed into Escherichia coli K-12-derived Origami cells for expression following the same conditions as in our previous work (for a more detailed description see de la Rosa et al 10 ). In short, expressed recombinant ScNtx, MlatA1 or r.D.H were submitted to a two-step purification process: by Ni-NTA (Ni-nitrilotriacetic acid) affinity chromatography according to the method of polyhistidine-tagged proteins user manual (Qiagen); and by reversed-phase high-performance liquid chromatography (RP-HPLC) (Agilent 1100 series; Agilent, CA) loading them onto an analytical C 18 column (4.6 × 250 mm 2 , VYDAC ® ).…”

Successful refolding and NMR structure of rMagi3: A disulfide‐rich insecticidal spider toxin

“…Since uncontrolled disulfide‐bond oxidative scramble of cysteine‐rich arachnid toxins could occur during bacterial expression, the trx‐6His‐rMagi3 fusion protein was submitted to a refolding process to improve the yield of a well‐structured rMagi3. After that, the trx‐6His‐rMagi3 was enzymatically cleaved with thrombin, and the trx‐His6‐LVPR residue was satisfactorily removed from the reaction mixture by a second IMAC separation [Fig.…”

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

“…To date, bacterial expression still remains the preferred system for the heterologous expression of toxins, especially for small and cysteine-less toxins like actinoporins from sea anemones ( Alegre-Cebollada et al, 2007 ). Bacterial expression has been successfully used to produce the majority of scorpion toxins produced so far ( Amorim et al, 2018 ), and it has been widely used to express snake toxins ( Clement et al, 2016 ; Shulepko et al, 2017 ; David et al, 2018 ; Guerrero-Garzón et al, 2018 ; Russo et al, 2019 ), conotoxins from cone snails ( Yu et al, 2018 ; Nielsen et al, 2019 ; Liu et al, 2020 ), and spider toxins ( Meng et al, 2011 ; Souza et al, 2012 ; Chassagnon et al, 2017 ; Wu et al, 2017 ). Nevertheless, as mentioned before, PTMs and notably, complex disulfide-bonding patterns pose a considerable challenge for the general use of bacterial expression systems.…”

“…In addition, the SHuffle ® strain expresses a periplasmic disulfide isomerase (DsbC) in the cytoplasm to enhance native disulfide-bond formation ( Lobstein et al, 2012 ). SHuffle ® and Rosetta-gami™ (an Origami™ derivative) strains have been employed in the recombinant production of venom peptides ( Li et al, 2006 ; Sermadiras et al, 2013 ; Clement et al, 2016 ). However, they tend to exhibit low growth and yield ( Nozach et al, 2013 ), and successful expression of correctly folded disulfide-rich peptides can require the co-expression of other chaperones ( Levy et al, 2001 ).…”