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Updated: Jun 27, 2026
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Published on: July 30, 2014
Bioinformatic Analysis and Recombinant Expression of the Stonustoxin β-Subunit for Polyclonal Antibody Development
Mohammadreza Rahmani1, Shahram Nazarian1, Hossein Samiei-Abianeh1,2
1Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.
Background:
Stonefish (Synanceia spp.) are among the most venomous marine organisms. Their venom contains stonustoxin (SNTX), a heterodimeric toxin that induces severe hemolytic and myotoxic effects primarily mediated by its β-subunit.
Objective:
To produce a recombinant SNTX β-subunit and develop neutralizing polyclonal antibodies against SNTX.
Methods:
A DNA cassette encoding immunogenic regions of the β-sntx was designed using bioinformatics analysis, and codon-optimized for expression in E. coli. The construct was cloned into pET17b vector, and expressed in E. coli BL21 (DE3). The recombinant protein was purified via Ni-NTA affinity chromatography. For antibody production, rabbits were immunized subcutaneously with the recombinant protein emulsified in Freund's complete adjuvant, followed by booster doses at 2-week intervals. Antiserum was purified using protein G chromatography, and antibody titers were assessed by indirect Enzyme-Linked Immunosorbent Assay (ELISA).
Results:
Epitope mapping revealed key immunogenic regions within residues 124-654 of the β-SNTX subunit. Following codon optimization, the codon adaptation index (CAI) increased to 0.93. Recombinant protein production was confirmed by SDS-PAGE and Western blot demonstrating successful purification of a 73 kDa recombinant protein (including TRX/His-tags), with a yield of 40 mg/L. Immunization of rabbits elicited a strong polyclonal IgG response, with antibody titers reaching 1:25,600 following the third booster. Purified IgG (1.8 mg/mL) exhibited high sensitivity in ELISA, detecting the recombinant β-SNTX at concentrations as low as 31.25 ng.
Conclusion:
The recombinant β-SNTX subunit demonstrated strong immunogenicity, inducing high-affinity antibodies with specific binding activity against the native toxin. The resulting antiserum demonstrated significant neutralization potential, highlighting its promise for antivenom development.
