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

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.