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Title: Botulinum Neurotoxin B Recognizes Its Protein Receptor With High Affinity And Specificity

Abstract

No abstract prepared.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Stanford Linear Accelerator Center (SLAC)
Sponsoring Org.:
USDOE
OSTI Identifier:
897754
Report Number(s):
SLAC-REPRINT-2006-191
TRN: US200705%%321
DOE Contract Number:
AC02-76SF00515
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nature 444:1092-1095,2006
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AFFINITY; PROTEINS; SPECIFICITY; Other,OTHER

Citation Formats

Jin, R.S., Rummel, A., Binz, T., Brunger, A.T., and /Howard Hughes Med. Inst. /SLAC, SSRL. Botulinum Neurotoxin B Recognizes Its Protein Receptor With High Affinity And Specificity. United States: N. p., 2007. Web.
Jin, R.S., Rummel, A., Binz, T., Brunger, A.T., & /Howard Hughes Med. Inst. /SLAC, SSRL. Botulinum Neurotoxin B Recognizes Its Protein Receptor With High Affinity And Specificity. United States.
Jin, R.S., Rummel, A., Binz, T., Brunger, A.T., and /Howard Hughes Med. Inst. /SLAC, SSRL. Tue . "Botulinum Neurotoxin B Recognizes Its Protein Receptor With High Affinity And Specificity". United States. doi:.
@article{osti_897754,
title = {Botulinum Neurotoxin B Recognizes Its Protein Receptor With High Affinity And Specificity},
author = {Jin, R.S. and Rummel, A. and Binz, T. and Brunger, A.T. and /Howard Hughes Med. Inst. /SLAC, SSRL},
abstractNote = {No abstract prepared.},
doi = {},
journal = {Nature 444:1092-1095,2006},
number = ,
volume = ,
place = {United States},
year = {Tue Jan 16 00:00:00 EST 2007},
month = {Tue Jan 16 00:00:00 EST 2007}
}
  • With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay,more » because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.« less
  • A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less
  • No abstract prepared.
  • Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level withmore » high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.« less
  • Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less