title: Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli.
abstract: BACKGROUND
  Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.
  
  
  METHODS
  The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.
  
  
  RESULTS
  Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.
  
  
  CONCLUSIONS
  The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method.
  
  
  CONCLUSIONS
  We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.
authors.affiliation: Department of Physiology and Pathophysiology, Friedrich-Alexander Universität Erlangen-Nürnberg, Universitätsstraße 17, 91054 Erlangen, Germany.
authors.forename: A O
authors.initials: AO
authors.lastname: O'Reilly
authors.forename: A R
authors.initials: AR
authors.lastname: Cole
authors.forename: J L S
authors.initials: JL
authors.lastname: Lopes
authors.forename: A
authors.initials: A
authors.lastname: Lampert
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012605:Scorpions / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: C115530:Bj-xtrIT toxin; D004220:Disulfides; D019476:Insect Proteins; D018832:Molecular Chaperones; D009498:Neurotoxins; D011994:Recombinant Proteins
keywords: Chaperones; Crystal structure; Disulfide-linked toxin; Protein expression; Protein folding; Synchrotron radiation circular dichroism spectroscopy