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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Botulinum Toxins, Type A / chemistry*
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Botulinum Toxins, Type A / genetics*
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Botulinum Toxins, Type A / isolation & purification
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Botulinum Toxins, Type A / metabolism
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Drug Carriers / chemistry
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Drug Carriers / isolation & purification
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Drug Carriers / metabolism
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Drug Delivery Systems / methods*
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Escherichia coli / genetics
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Mice
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Motor Neurons / cytology
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Motor Neurons / metabolism
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Neuromuscular Junction / cytology
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Neurons / cytology*
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Neurons / metabolism*
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Protein Engineering
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Protein Structure, Tertiary
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Protein Transport
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / genetics*
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Reflex / drug effects
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Toes / physiology
Substances
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Drug Carriers
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Recombinant Fusion Proteins
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Botulinum Toxins, Type A