Functional expression and genetic alteration of an alpha scorpion neurotoxin.
The alpha neurotoxin Lqh alpha IT is toxic to both insects and mammals but exhibits a bioactivity ratio favoring insects (insect/mammal approximately 2). With the objective of increasing this ratio by genetic manipulation of the amino acid sequence, a cDNA clone encoding Lqh alpha IT was used to produce recombinant variants of the toxin in a high efficiency bacterial expression system. The unmodified recombinant toxin, isolated from inclusion bodies and renatured in vitro, exhibited chemical and biological properties indistinguishable from those of the authentic native toxin. Alteration of the toxin by site-directed mutagenesis led to a substantial reduction in anti-mammalian toxicity (mouse LD50 reduced 6.4-fold) but only a slight reduction (x 1.5) in the insect ED50 value for paralysis. The reduction in anti-mammalian toxicity was correlated with a approximately 2-fold reduction of its potency for slowing of sodium channel inactivation in mammalian neurons, while no change in mutant toxin binding affinity to insect neuronal receptors was registered. These results demonstrate for the first time expression of a recombinant sodium channel neurotoxin in Escherichia coli and the use of site-directed mutagenesis to improve phylogenetic selectivity. This recombinant approach provides a promising strategy for optimizing the selective toxicity of peptide neurotoxins.
Zilberberg
N
N
Department of Botany, Faculty of Life Sciences, Tel-Aviv University, Israel.
Gordon
D
D
Pelhate
M
M
Adams
M E
ME
Norris
T M
TM
Zlotkin
E
E
Gurevitz
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000648:ultrastructure; D005727:Ganglia, Spinal / Q000502:physiology; D007313:Insecta; D007544:Isopropyl Thiogalactoside; D008322:Mammals; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D009498:Neurotoxins / Q000737:chemistry / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000633:toxicity; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000633:toxicity; D012605:Scorpions; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015222:Sodium Channels / Q000187:drug effects; D013045:Species Specificity
D009498:Neurotoxins; D011994:Recombinant Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; D007544:Isopropyl Thiogalactoside