Expression of functional recombinant scorpion beta-neurotoxin Css II in E. coli.
The gene for a beta-neurotoxin [Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [(3)H]GABA [gamma-aminobutyric acid (GABA)] in neuronal cells as effectively as natural beta-toxins.
Johnson
T M
TM
Department of Biochemistry & Molecular Genetics, CH19 Room B31, University of Alabama at Birmingham, Birmingham, AL 35294-2041, USA.
Quick
M W
MW
Sakai
T T
TT
Krishna
N R
NR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D001681:Biological Assay; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis*; D030162:Reptilian Proteins; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D009498:Neurotoxins; D011994:Recombinant Proteins; D030162:Reptilian Proteins; D012604:Scorpion Venoms; C066386:scorpion toxin Css II; C053969:scorpion toxin II, Androctonus