Expression, purification and comparative characterisation of enzymatically deactivated recombinant botulinum neurotoxin type A

Yang, Weiping; Lindo, Paul; Riding, Stephen; Chang, Tzuu Wang; Cai, Shuowei; Van, Thuan; Kukreja, Roshan; Zhou, Yu; Vasa, Kruti; Singh, Bal Ram

doi:10.1504/tbj.2008.026477

Cited by 18 publications

(12 citation statements)

References 32 publications

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“…Recombinant BoNT/A LC and DR BoNT/A were isolated and purified from Escherichia coli transformed with pBN3 vectors harboring BoNT/A LC and the double mutant DR BoNT/A genes respectively, as described previously [25,26]. BoNT/A, recombinant BoNT/ B & E LC were provided by BB Tech (North Dartmouth, MA).…”

Section: Methodsmentioning

confidence: 99%

“…DR BoNT/A is the recombinant BoNT/A with two mutations on the enzymatic site of light chain (E224A/E262A). This detoxified protein has been confirmed to have identical structure and biological functions (binding, internalization and translocation on neuronal cells) to its wild type toxin, with much reduced endopeptidase activity [25]. Due to the absence of endopeptidase activity, the toxicity of DR BONT/A is approximately 100,000 fold less than that of native toxin, therefore providing an excellent surrogate to study BoNT without sophisticated laboratory requirements for tier one select agents.…”

Section: Introductionmentioning

confidence: 99%

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RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

Janardhanan

Mello

Singh

et al. 2013

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A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 minutes. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91% to 116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

Janardhanan

Mello

Singh

et al. 2013

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“…However, both methods involve the use of C. botulinum culture which require regulatory issues, including a facility to address biosafety concerns. As an alternative approach, we have utilized a PCR amplified DNA fragment encoding LC-TD/A region from the DrBoNT/A clone, which is a non-toxic variant of BoNT/A, previously produced in our lab [17]. The method described in the current paper involves use of heterologous expression E.coli system for production of rLC-TD/A protein which resulted in 2 to 20-fold higher yield (1.5 mg per gm cell paste or 1.5 mg per liter of E.coli culture) with > 95 % purity as compared to the methods published (20 fold higher than that reported by [15], and 2 fold higher than that by [16], respectively).…”

Section: Discussionmentioning

confidence: 99%

“…The DNA fragment for rLC-TD/A was obtained from deactivated recombinant BoNT/A (DrBoNT/A) vector clone [17] that is exempted by CDC from the select agent regulations. DrBoNT/A clone produces catalytically inactive protein consisting of two glutamic acid mutations (E224 and E262) to alanine at the active site of BoNT/A LC which has been demonstrated by in vitro SNAPtide® [17] and mouse neuromuscular junction assay [18].…”

Section: Methodsmentioning

confidence: 99%

“…DrBoNT/A clone produces catalytically inactive protein consisting of two glutamic acid mutations (E224 and E262) to alanine at the active site of BoNT/A LC which has been demonstrated by in vitro SNAPtide® [17] and mouse neuromuscular junction assay [18]. Primers were designed for the amplification of rLC-TD/A region from DrBoNT/A according to the BoNT/A sequence in the GenBank database under Accession No.…”

Section: Methodsmentioning

confidence: 99%

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High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

et al. 2017

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Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain and heavy chain. The light chain is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the heavy chain are required for cell binding, and translocation of light chain across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2 to 20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in Escherichia coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify >95 % pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and rLC/A (recombinant light chain A) suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.

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A Highly Active and Selective Nanocomposite Catalyst for C₇₊ Paraffin Isomerization

Ying

2006

Angewandte Chemie

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Ein Pt/WO3/Al‐ZrOx‐Nanokomposit wandelt verschiedene Kohlenwasserstoffe bereits bei 150 °C effizient um (siehe Bild, X=Umsatz), ohne dass starkes Cracken oder Verkokung auftreten. Der neuartige Katalysator könnte zur Produktion von Treibstoffen mit hoher Oktanzahl bei niedrigem Arengehalt genutzt werden, da er n‐Alkane mit ausgezeichneter Selektivität in mehrfach verzeigte Isomere überführt.

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Expression, purification and comparative characterisation of enzymatically deactivated recombinant botulinum neurotoxin type A

Cited by 18 publications

References 32 publications

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

A Highly Active and Selective Nanocomposite Catalyst for C₇₊ Paraffin Isomerization

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Expression, purification and comparative characterisation of enzymatically deactivated recombinant botulinum neurotoxin type A

Cited by 18 publications

References 32 publications

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

A Highly Active and Selective Nanocomposite Catalyst for C7+ Paraffin Isomerization

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A Highly Active and Selective Nanocomposite Catalyst for C₇₊ Paraffin Isomerization