A comparative study of the performance of E. coli and K. phaffii for expressing α-cobratoxin

• Published in: Toxicon (Oxford) 2024-02, Vol.239, p.107613-107613, Article 107613
• Main Authors: Damsbo, Anna, Rimbault, Charlotte, Burlet, Nick J., Vlamynck, Anneline, Bisbo, Ida, Belfakir, Selma B., Laustsen, Andreas H., Rivera-de-Torre, Esperanza
• Format: Article
• Language: English
• Subjects: Bacterial expression; Cobra Neurotoxin Proteins - chemistry; Cobra Neurotoxin Proteins - metabolism; Elapid Venoms - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Receptors, Nicotinic - metabolism; Recombinant toxin expression; Snake venom; Three Finger Toxins; Toxins, Biological; Venoms; Yeast expression; α-cobratoxin
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2024.107613

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs. [Display omitted] •α-cobratoxin produced in bacteria or yeast is not completely identical to the native toxin structure•α-cobratoxin produced in yeast has a similar circular dichroism spectrum as the native toxin and binds the target receptor•Solubilization tags aid in enhancing the expression of soluble protein, but removal negatively affects net yield•E. coli and K. phaffii-expressed α-cobratoxin retains functional binding to its target receptor.•Recombinant toxins can be used to advance antivenom research and the development of toxin-derived therapeutics.