4kyp

Beta-Scorpion Toxin folded in the periplasm of E.coli

spinqualitylabelsall models 4kyp, resolution 1.70Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 4kyp is a 4 chain structure with sequence from Hottentotta judaicus. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.7Å Ligands: PG4, PGE Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed 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 beta-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. GENERAL SIGNIFICANCE: 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. Chaperone-mediated native folding of a beta-scorpion toxin in the periplasm of Escherichia coli.,O'Reilly AO, Cole AR, Lopes JL, Lampert A, Wallace BA Biochim Biophys Acta. 2014 Jan;1840(1):10-5. doi: 10.1016/j.bbagen.2013.08.021., Epub 2013 Aug 30. PMID:23999087 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

Proteopedia Page Contributors and Editors (what is this?)

OCA