Three-Finger Viper Toxins: cDNA Cloning and Expression in E. coli Using a Chimeric (Hybrid) Construction with a SUMO Fusion Partner Protein

Abstract— Objective: Three-finger toxins (TFTs) form one of the most abundant families of toxins in snake venoms. TFTs are characteristic of most elapid venoms, but have almost never been found in viper venoms. The aim of this work was to obtain viper TFTs, the mRNAs of which are present in the viper venom glands, and to study their properties. Methods: PCR was used to amplify cDNA encoding TFTs from viper venom glands. The TFTs were then heterologously expressed in E. coli as plant SUMO fusion proteins, purified by affinity chromatography, and cleaved using the plant protease BdSENP1, followed by final chromatographic purification. Results and Discussion: Using the venom glands of the vipers Vipera nikolskii and V. berus, 21 cDNA clones encoding this group of toxins were obtained. The amino acid sequences of nine TFTs were deduced from their corresponding cDNA sequences. All viper TFTs belong to the group of nonconventional toxins, and their sequences contain 9 cysteine residues. The TFT encoded by one of the transcripts was obtained. Analysis of its biological activity showed that this toxin is a weak antagonist of neuronal nicotinic acetylcholine receptors of the α7 and α3β2 subtypes. Using a SUMO fusion protein approach, an attempt was made to obtain the TFT Aze-2 of the viper Azemiops feae, which was identified in minimal quantities in the venom of this snake. However, this method failed to yield a toxin matching the exact mass of Aze-2. Conclusions: As a result of the work, the amino acid sequences of 9 viper TFTs were established, one of which was obtained by gene expression in E. coli cells and showed the ability to interact with neuronal nicotinic acetylcholine receptors of the α7 and α3β2 subtypes.