Recombinant expression and insecticidal properties of a Conus ventricosus conotoxin-GST fusion protein.

摘要

A novel conotoxin, conotoxin Vn2, was recently isolated from the venom of Conus ventricosus, a worm-hunting cone snail species living in the Mediterranean Sea. Analysis of conotoxin Vn2 amino acid sequence suggested that it is a member of the O1 superfamily of conotoxins. Conotoxin Vn2 displays quite a high degree of sequence similarity with bioactive peptides targeting calcium channels and in particular with the omega conotoxin PnVIB, extracted from the venom of the molluscivorous cone snail Conus pennaceus. In this work we describe the development of a heterologous expression system to obtain a glutathione-S-transferase (GST) fusion product of conotoxin Vn2 in a pure form and in a sufficient amount to characterize its bioactivity. The fusion product has been expressed in recombinant form in Escherichia coli cells, purified, and its neurotoxic activity has been assayed on the larvae of the moth Galleria mellonella, a simple experimental model to test the toxicity of compounds in insects. Moreover the conotoxin Vn2 Asp2His mutant has been produced to analyse the role of this aspartic acid residue in the toxin bioactivity, as an acidic amino acid is conserved in this position in all the O1 superfamily C. ventricosus conotoxins. Results obtained indicate that indeed conotoxin Vn2 has strong insecticidal properties at a dose of only 100 pmol/g of body weight. Surprisingly, mutation of Asp2 to His leads to enhanced toxicity in the larvae model system opening up interesting possibilities for the use of conotoxin Vn2 variants in environmental friendly crop protection applications.