Production of active, insect-specific scorpion neurotoxin in yeast.
A cDNA encoding the Androctonus australis Hector insect toxin 1 (AaH IT1) was expressed in yeast leading to secretion of fully biologically active protein. Three different multicopy plasmids were constructed using PCR. Expression was directed by the strong PGK1 promoter of the yeast 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 yeast alpha-mating-factor precursor; the pBAL 7-alpha-KREAEA-AaH IT1 includes the cDNA sequence encoding the KR(EAEA) processing sequence of the alpha-mating factor, and pBAL 7-alpha-KR-AaH IT1 encodes the KR fused directly to the AaH IT1 gene. The yeast alpha-mating-factor signal peptide launched the pro-alpha-mating-factor-AaH IT1 fusion protein into the secretory pathway. The fusion proteins are expected to be cleaved in the Golgi by the KEX2 endopeptidase and the STE13 dipeptidyl aminopeptidase, leading to release of mature AaH IT1. Pulse/chase labelling of transformed yeast protoplasts, followed by SDS/PAGE analysis of proteins immunoprecipitated from either the lysate or the extracellular fluid, showed that AaH IT1 was produced. The highest concentration of recombinant AaH IT1 in the culture medium, as determined using a 125I-AaH IT1 specific radioimmunoassay, was 4 micrograms/l (0.5 nM). The recombinant toxin was fully biologically active against cockroaches as assessed by injection and comparison to native AaH IT1. Moreover, it competed with radiolabelled native toxin for its receptor on the voltage-sensitive Na+ channel with a dissociation constant of 0.5 nM.
Martin-Eauclaire
M F
MF
Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark.
Søgaard
M
M
Ramos
C
C
Cestèle
S
S
Bougis
P E
PE
Svensson
B
B
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003058:Cockroaches / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D009498:Neurotoxins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism / Q000633:toxicity; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D015222:Sodium Channels / Q000187:drug effects
D018076:DNA, Complementary; D009479:Neuropeptides; D009498:Neurotoxins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; C064067:scorpion toxin AaH IT1