In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli.
The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E. coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.
Turkov
M
M
Department of Plant Sciences, Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Israel.
Rashi
S
S
Noam
Z
Z
Gordon
D
D
Ben Khalifa
R
R
Stankiewicz
M
M
Pelhate
M
M
Gurevitz
M
M
D000818:Animals; D001369:Axons / Q000187:drug effects; D017123:Bacteriophage T7 / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003058:Cockroaches / Q000187:drug effects; D004175:Diptera / Q000187:drug effects / Q000254:growth & development; D004355:Drug Stability; D004926:Escherichia coli; D017344:Genes, Insect; D005814:Genes, Viral; D015640:Ion Channel Gating / Q000187:drug effects; D007814:Larva; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D010243:Paralysis / Q000139:chemically induced; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C076349:LqhIT2 insect toxin; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms