ABSTRACT
The long half-life of botulinum neurotoxin serotype A (BoNT/A) in cells poses a challenge in developing post-exposure therapeutics complementary to existing antitoxin strategies. Delivery vehicles consisting of the toxin heavy chain (HC), including the receptor-binding domain and translocation domain, connected to an inhibitory cargo offer a possible solution for rescuing intoxicated neurons in victims paralyzed from botulism. Here, we report the expression and purification of soluble recombinant prototype green fluorescent protein (GFP) cargo proteins fused to the entire BoNT/A-HC (residues 544-1295) in Escherichia coli with up to a 40 amino acid linker inserted between the cargo and BoNT/A-HC vehicle. We show that these GFP-HC fusion proteins are functionally active and readily taken up by cultured neuronal cells as well as by neuronal cells in mouse motor nerve endings.
Subject(s)
Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/genetics , Drug Delivery Systems/methods , Neurons/cytology , Neurons/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Animals , Botulinum Toxins, Type A/isolation & purification , Botulinum Toxins, Type A/metabolism , Drug Carriers/chemistry , Drug Carriers/isolation & purification , Drug Carriers/metabolism , Escherichia coli/genetics , Mice , Motor Neurons/cytology , Motor Neurons/metabolism , Neuromuscular Junction/cytology , Protein Engineering , Protein Structure, Tertiary , Protein Transport , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Reflex/drug effects , Toes/physiologyABSTRACT
The light chain of botulinum neurotoxin A (BoNT/A-LC) is a zinc-metalloprotease that requires two extended exosites for optimal substrate binding and recognition of its intracellular target SNAP25. CFP and YFP connected through SNAP25 peptide (141-206) containing both exosites (CsY) has been used in a FRET-based assay for BoNT/A. To further improve the FRET efficiency in this BoNT/A substrate for in vitro high-throughput assays, we explored the feasibility of enhancing the capture of CFP emission by doubling the number of YFP acceptors. In comparison to CsY, the tandem fluorescence substrates CsYY and YsCsY enhanced the ratiometric fluorescence signal between YFP and CFP. YsCsY, containing two substrate sites, offered the greatest fluorometric change upon toxin-catalyzed cleavage. In addition to known approaches for enhancing fluorescence yield through various mutations, this alternative tandem substrate approach can boost the FRET signal and is particularly useful for substrates requiring extensive exosite recognition for specificity.
