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 simi lar to 2). With the objective of increasing this ratio by genetic mani pulation of the amino acid sequence, a cDNA clone encoding Lqh alpha I T was used to produce recombinant variants of the toxin in a high effi ciency bacterial expression system. The unmodified recombinant toxin, isolated from inclusion bodies and renatured in vitro, exhibited chemi cal and biological properties indistinguishable from those of the auth entic native toxin, Alteration of the toxin by site-directed mutagenes is led to a substantial reduction in anti-mammalian toxicity (mouse LD (50) reduced 6.4-fold) but only a slight reduction (x1.5) in the insec t ED(50) value for paralysis. The reduction in anti-mammalian toxicity was correlated with a similar to 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 w as registered. These results demonstrate for the first time expression of a recombinant sodium channel neurotoxin in Escherichia coli and th e use of site-directed mutagenesis to improve phylogenetic selectivity . This recombinant approach provides a promising strategy for optimizi ng the selective toxicity of peptide neurotoxins.