Immunogenic potential and neutralizing ability of a heterologous version of the most abundant three-finger toxin from the coral snake Micrurus mipartitus

Abstract

Background: Micrurus mipartitus is a coral snake of public health concern in Colombia. Its venom is mainly composed of three-finger toxins (3FTxs), Mipartoxin-1 being the most abundant protein partially responsible for its lethal effect. In this work, we present the production of Mipartoxin-1 in a recombinant form and evaluate its immunogenic potential. Methods: A genetic construct HisrMipartoxin-1 was cloned into the pET28a vector and heterologous expression was obtained in E. coli BL21 (DE3). The recombinant HisrMipartoxin-1 protein was extracted from inclusion bodies, refolded in vitro, and isolated by affinity and RP-HPLC chromatography. The lethal effect of HisrMipartoxin-1 was tested, and antibodies against HisrMipartoxin-1 were produced by immunization in rabbits. The antibody titers were monitored by an ELISA test. The neutralizing ability of the antibodies, against the lethal effect of native toxins and M. mipartitus venom, was also assessed. Results: HisrMipartoxin-1 was detected on SDS-PAGE, with a molecular mass of around 11 kDa. The retention time was 16.0 minutes. HisrMipartoxin-1 did not exhibit lethality in mice; however, antibodies against HisrMipartoxin-1 recognized the native toxin, the whole venom of M. mipartitus, and a 3FTx from another species within the Micrurus genus. Furthermore, antibodies against HisrMipartoxin-1 completely neutralized the lethal effect of native Mipartoxin-1 in mice but not M. mipartitus whole venom. Conclusion: These findings indicate that HisrMipartoxin-1 might be used as an immunogen to develop anticoral antivenoms or complement them. This work is the first report of the heterologous expression of 3FTx from M. mipartitus.

Keywords: Antibodies; Coral snake antivenoms; Micrurus mipartitus; Mipartoxin-1; Recombinant protein; Three-finger toxin.

Figure 1.. HisrMipartoxin-1 and digestion into pET28a. (A) Diagram of the genetic construction HisrMipartoxin-1. (B) Cleavage pET28a with XhoI and EcoRV on 1% agarose gel stained with ethidium bromide (Sigma, Saint Louis, MO, USA). MW: molecular weight marker (1 kb Plus DNA Ladder) (NEB); 1, 2, and 3: individual HisrMipartoxin-1 clones. The arrow indicates the 1578 bp expected fragment containing the 280 bp target insert.

Figure 2.. HisrMipartoxin-1 expressed in E. coli BL21 (DE3) strain. (A) SDS-PAGE analysis of HisrMipartoxin-1 before (at 0 hours) and after IPTG induction (at 2 and 8 hours). M: molecular mass marker; T: total protein; S: soluble fraction; I: insoluble fraction. (B) In vitro refolding and isolation of HisrMipartoxin-1. IBs: solubilized inclusion bodies; RP: refolded protein; FT: Flow-through; W1 and W2: Wash 1 and 2; E: Elution. (C) Cleavage of 6His-tag with TEV protease. rMipartoxin-1 was detected as a band of about 8 kDa (black arrow). HisrMipartoxin-1 showed approximately 11 kDa (dashed arrow). (D) Western blot analysis using the anti-Mipartoxin-1 antibody. Lane 1: The M. mipartitus venom profile contained different proteins; the region around 10 kDa is enriched in 3FTxs, particularly Mipartoxin-1 (7 kDa). The intensity of the band is due to their abundance in the M. mipartitus proteome. Lane 2: The molecular mass of HisrMipartoxin-1 was approximately 11 kDa (arrow). M: molecular mass marker in kDa.

Figure 3.. Purification by RP-HPLC chromatography. HisrMipartoxin-1 eluted at 16.0 min. An acetonitrile linear gradient and 1 mL/min flow were applied for elution.

Figure 4.. Antibody titers by ELISA in serum (average of four bleeding) against HisrMipartoxin-1. Sera from four bleedings in different dilutions (1:10 to 1:10000) were used, and absorbances were recorded at 490 nm. * Indicates differences with the pre-immune serum (p < 0.0001). Data correspond to the mean ± SD (n = 3).

Figure 5.. IgG purification, immunorecognition, and anti-HisrMipartoxin-1-IgG titers. (A) Fractionation IgG of hyperimmune serum using caprylic acid. Efficiency analysis was made by 10% SDS-PAGE under non-reducing conditions. The gels-stained with Coomassie Blue R-250. M: broad range molecular marker (kDa). 1: anti-HisrMipartoxin-1 serum; 2: IgG obtained after fractionation; 3: Albumin standard. The dashed arrow indicates the IgG band, and the black arrow indicates albumin. (B) Immunorecognition by ELISA of anti-HisrMipartoxin-1-IgG from each bleed (bleedings 1 to 4), and pre-immune serum (PI) against HisrMipartoxin-1. A 96-well plate was coated with HisrMipartoxin-1, and IgG from each bleeding was used at a 1:100 dilution (from an initial concentration of 50 mg/mL). The pre-immune serum was used at the same dilution. (C) Titration curve of anti-HisrMipartoxin-1-IgG by ELISA against HisrMipartoxin-1. IgG from four bleedings was used in dilutions from 1:10 to 1:10000 (from an initial concentration of 50 mg/mL). For B and C, * indicates differences compared to the pre-immune serum (p < 0.0001). Data correspond to the mean ± SD (n = 3).

Figure 6.. Immunoreactivity by ELISA of sera and IgG anti-HisrMipartoxin-1 against M. mipartitus venom (V-Mmip), Mipartoxin-1 (native), HisrMipartoxin-1, and Clarkitoxin-I-Mdum. *Indicates differences when compared to the pre-immune serum (p < 0.0001). Data correspond to the mean ± SD (n = 3).