PRODUCTION OF ACTIVE, INSECT-SPECIFIC SCORPION NEUROTOXIN IN YEAST

A cDNA encoding the Androctonus australis Hector insect toxin 1 (AaH I T1) was expressed in yeast leading to secretion of fully biologically active protein. Three different multicopy plasmids were constructed us ing PCR. Expression was directed by the strong PGK1 promoter of the ye ast vector pMA 91. Plasmid pMA 91-AaH IT1 encodes AaH IT1 and its own signal peptide. In the two other constructions, the cDNA encoding the mature part of AaH IT1 is fused to the prepro-signal sequence of the y east alpha-mating-factor precursor; the pBAL 7-alpha-KREAEA-AaH IT1 in cludes the cDNA sequence encoding the KR(EAEA) processing sequence of the alpha-mating factor, and pBAL 7-alpha-KR-AaH IT1 encodes the KR fu sed directly to the AaH IT1 gene. The yeast alpha-mating-factor signal peptide launched the pro-alpha-mating-factor-AaH IT1 fusion protein i nto the secretory pathway. The fusion proteins are expected to be clea ved in the Golgi by the KEX2 endopeptidase and the STE13 dipeptidyl am inopeptidase, leading to release of mature AaH IT1. Pulse/chase labell ing of transformed yeast protoplasts, followed by SDS/PAGE analysis of proteins immunoprecipitated from either the lysate or the extracellul ar fluid, showed that AaH IT1 was produced. The highest concentration of recombinant AaH IT1 in the culture medium, as determined using a I- 125-AaH IT1 specific radioimmunoassay, was 4 mu g/l (0.5 nM). The reco mbinant toxin was fully biologically active against cockroaches as ass essed by injection and comparison to native AaH IT1. Moreover, it comp eted with radiolabelled native toxin for its receptor on the voltage-s ensitive Na+ channel with a dissociation constant of 0.5 nM.