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

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Resumo

Three-finger toxins (TFTs) form one of the most abundant families of toxins in snake venoms. TFTs are common for most Elapid venoms, but are almost never found in viper venoms. 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 9 TFTs were deduced from the obtained cDNA sequences. The identified sequences have signal peptides containing 19-21 amino acid residues, followed by a mature protein consisting of 67 residues. All viper TFTs belong to the group of non-conventional toxins, and their sequences contain 9 cysteine residues. The TFT encoded by one of the transcripts was obtained by heterologous expression in E. coli cells as a fusion protein with the plant partner protein SUMO, followed by cleavage with the specific plant protease BdSENP1 and chromatographic purification. The structure of the obtained protein was confirmed by mass spectrometry. Analysis of its biological activity showed that this toxin is a weak antagonist of nicotinic acetylcholine receptors of the neuronal α7 and α3β2 subtypes. Using the fusion protein with SUMO, we also attempted to obtain the TFT Aze-2 of the viper Azemiops feae, the amino acid sequence of which was previously established by us as a result of transcriptome analysis of the venom gland of A. feae, and the protein itself was identified in minimal quantities in the venom of this snake. However, a toxin exactly corresponding in mass to Aze-2 could not be obtained using this approach. Thus, 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 nicotinic acetylcholine receptors of the neuronal α7 and α3β2 subtypes.

Sobre autores

D. Sukhov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; MIREA – Russian Technological University

Moscow, Russia; Moscow, Russia

L. Ojomoko

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

I. Shelukhina

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

M. Vladykina

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

V. Kost

Weizmann Institute of Science

Department of Chemical and Structural Biology Rehovot, Israel

R. Ziganshin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

O. Geraskina

M.V. Lomonosov Moscow State University, Faculty of Biology

Moscow, Russia

S. Balandin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

T. Ovchinnikova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

V. Tsetlin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

Yu. Utkin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yutkin@yandex.ru
Moscow, Russia

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