Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B

Abstract

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosisin neuronal and non-neuronal cells due to their peptidase activity on exocytic isoformsof SNARE proteins. They are produced by Clostridia as single-chain polypeptides that areproteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds toheavy, targeting domains. This format of two subunits linked by disulfide bonds is requiredfor the full neurotoxicity of BoNTs.We have generated a recombinant version of BoNT/B thatconsists of the light chain of the toxin fused to the protein transduction domain of the humanimmunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC,expressed in Escherichia coli and purified by affinity chromatography, penetrated membranesand exhibited strong enzymatic activity, as evidenced by cleavage of the SNAREsynaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack ofsynaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The noveltool reported herein disrupts the function of a SNARE protein within minutes in cells thatmay or may not express the receptors for the BoNT/B heavy chain, and without the needfor transient transfection or permeabilization.