Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro.
The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.
Estrada
Georgina
G
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM. Apartado Postal 510-3, Cuernavaca Morelos, 62250, Mexico.
Garcia
Blanca I
BI
Schiavon
Emanuele
E
Ortiz
Ernesto
E
Cestele
Sandrine
S
Wanke
Enzo
E
Possani
Lourival D
LD
Corzo
Gerardo
G
D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006639:Histidine / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000378:metabolism; D007274:Injections, Intraperitoneal; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism; D014162:Transfection
C035413:Centruroides toxin; D004220:Disulfides; D009498:Neurotoxins; D011994:Recombinant Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; D007544:Isopropyl Thiogalactoside; D006639:Histidine