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Page 625 out of 632

FRET Peptide Substrates for the Botulinum Toxins Type A, B, and E and for Anthrax Lethal Factor.

Shine, N., Crawford, K.
Product: SNAPtide® Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin

The following FRET peptides have been designed at List Biological Laboratories as substrates for the botulinum toxin enzymes. SNAPtide®, Product #520 and #521 (U.S. Patent, No. 6,504,006 ) is readily recognized and cleaved by the Botulinum toxin type A (BTA), Product #130, and the BTA light chain (LcA), Product #610A. One of the substrates, Product #520, contains an oAbz/DNP FRET pair and the other, Product #521, a FITC/DABCYL FRET pair. Representative data are given below. VAMPtide®, Product #540 is readily recognized and cleaved by the Botulinum toxin type B light chain (LcB), Product #620A. This FRET substrate contains an oAbz/DNP FRET pair. SNAP Etide™, Product # 550 is readily recognized and cleaved by the Botulinum toxin type E light chain (LcE), Product #635A. This FRET substrate contains an oAbz/DNP FRET pair.

(LF) is the enzymatic component of anthrax lethal toxin which specifically cleaves the MAPK-kinase proteins. LF is also an ideal target for therapeutic inhibitors. FRET substrates for LF, MAPKKide®, Product #530 and 531, have been designed at List Biological Laboratories. One of the substrates, Product #530, contains an oAbz/DNP FRET pair and the other, Product #531, a FITC/DABCYL FRET pair. Product #531 is especially well suited for high throughput screening for the IC50 and inhibitor modality of potential inhibitors.  ...

SNAP-Etide®, a FRET Substrate for Botulinum Toxin Type E

Shine, N.; Christian, T.; Primak, Y.
Product: SNAP Etide® Peptide Substrate (o-Abz/Dnp)for C. botulinum Type E Neurotoxin

Materials - 

SNAP-EtideTMsubstrate (Product #550) and botulinum neurotoxin type E light chain, recombinant (Product #635A), are both products of List Biological Laboratories, Inc.

Methods - 

Fluorimentric Assay:

Continuous assays were performed on a SPECTRA max GEMINI XS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) using Greiner FLUO-TRAC black flat-bottomed plates (E&K Scientific, Campbell, CA).  Stock solutions of the FRET substrate was made in dimethylsulfoxide (DMSO).  Final dilutions were made in the appropriate buffer.  Plates were equilibrated at 37°C for 15 min prior to initiation of the reaction.  For all experiments the time-dependent increase in fluorescence intensity was monitored at 37°C. The excitation wavelength was set to 321 nm and emission to 418 nm. 

Buffer Optimization:

FRET assays were performed to test the activity of LcE with SNAP-EtideTMas a function of pH, Tween-20 and ZnCl2.  Three separate experiments were performed (Figure 1).  The cleavage reaction was initiated with addition of 5 nM LcE to the wells containing 10 µM SNAP-EtideTM in the appropriate buffer.  Initial velocities of cleavage in RFU/sec were evaluated and compared for each assay in order to determine the optimum buffer conditions for the reaction.

LcE Titration:

LcE titration experiment was performed in 50 mMHEPES, pH 7.8, 0.1% Tween-20, using 10 µM SNAP-EtideTMLcE was prepared at 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 µM concentrations.  Following equilibration, the cleavage reaction was initiated with addition of 10 µM SNAP-EtideTM.  Initial velocities of cleavage were plotted against LcE concentration (Figure 2).

Trypsin Digest: 

Dilutions of SNAP-EtideTMwere prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve 70, 60, 50, 40, 30, 25, 15, 7.5, 3.75, 1.88, and 0.94 µM concentrations.  The reaction was initiated with addition of 10 nM trypsin into each well.  End point readings were taken after 50 min.  A second round of 10 nM trypsin was added to each well in order to achieve total enzyme digestion.  The maximum fluorescence reached was graphed as RFU/5000 against SNAP-EtideTM concentration (Figure 3A).  An identical experiment was run using 2.5 nM LcE for digestion of SNAP-EtideTM.  Initial velocities of cleavage were graphed in RFU/sec against substrate concentration (Figure 3B).

Inner Filter Effect Correction: 

Dilutions of SNAP-EtideTM were prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve concentrations ranging from 250 µM to 2 µM.  Fluorescence end point readings of SNAP-EtideTM at each concentration were recorded.  In order to determine theinner filter effect at each substrate concentration another set of end point fluorescence (RFU) readings were recorded after addition of 5.0 µM free o-Abz-Lys.  Fluorescence intensity obtained for SNAP-EtideTM was then subtracted from the fluorescence intensity obtained for SNAP-EtideTMand o-Abz-Lysin order to obtain fluorescence for the free o-Abz-Lyspeptide.  The decrease in fluorescence of the o-AbzLysin the presence of SNAP-EtideTM reflects the inner filter effect (Table 1).  A correction factor is obtained for each SNAP-EtideTM concentration: 

correction factor = RFU (o-Abz-Lys) at each [SNAP-EtideTM] RFU (o-Abz-Lys)

Initial reaction rates were obtained for each substrate concentration after addition of 2.5 nM LcE.  The rates were corrected as given in Table 1.  The plots of initial velocity versus SNAP-EtideTM concentration (Figure 4)indicates a decreasing rate of cleavage at concentrations of substrate greater then 100 µM.  This is consistent with substrate inhibition.  The kinetic data was analyzed using the substrate inhibition equation from Kaleida Graph software: 

ax b+(x(1+x/c)) , where a = Vmax, b = Km, and c = Ki, competitive inhibition constant

Cationic polyamines inhibit anthrax lethal factor protease

Goldman ME, Cregar L, Nguyen D, Simo O, O'Malley S, Humphreys T
Product: Anthrax Lethal Factor (LF),Recombinant from B. anthracis

Lethal Factor protease assay:

Lethal Factor (20 nM final concentration) and MAPKK substrate (MAPKKide® 12.5 µM, final concentration) were purchased from List Biological Laboratories, Campbell, CA and used according to the fluorescence resonance energy transfer (FRET) method. ...

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

 

PubMed ID: 16762077

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Mabry R, Brasky K, Geiger R, Carrion R Jr, Hubbard GB, Leppla S, Patterson JL, Georgiou G, Iverson BL
Product: Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Sandwich ELISAs for LF detection:

Recombinant PA83 and PA63 were purchased from List Laboratories (New Jersey). PA63 is a cleavage product that is capable of binding LF (45). For the sandwich ELISA, 50 μl of PA at 63 kDa and 83 kDa (6 μg/ml) was applied to a 96-well plate and blocked with 2% milk-PBS as described above. For initial assays, LF was diluted in PBS or human serum at 5 μg/ml. For assays detecting LF in infected animals, serum was added to the plate initially diluted 1:1 in 2% milk-PBS and then serially diluted across the plate in duplicate. After a 1-h incubation, the plate was washed as described above. Goat anti-LF polyclonal serum (List Labs) was diluted 1:1,000 in 2% milk-PBS and added to the plate for a 1-h incubation in duplicate. The plate was then washed, followed by the addition of goat anti-rabbit IgG-HRP conjugate (Bio-Rad) diluted in 2% milk-PBS for 1 h. ELISA reactions were developed with OPD tablets (Sigma) and quenched by the addition of 50 μl of 4.5 M H2SO4. ...

PubMed ID: 16760326

Anthrax Lethal Toxin Has Direct and Potent Inhibitory Effects on B Cell Proliferation and Immunoglobulin Production

Fang H, Xu L, Chen TY, Cyr JM, Frucht DM
Product: Anthrax PA 63 - FITC Conjugate

Reagents and antibodies:

Recombinant anthrax PA and LF were purchased commercially and were stored in 1:1 glycerol-water at −20°C (List Biological Laboratories) for in vitro studies. Unless otherwise indicated, anthrax LT was administered in excess at concentrations of 2.5 μg/ml PA and 1 μg/ml LF. In selected experiments a proteolytically inactive mutant of LF was used as a negative control (E687C substitution in zinc binding site that eliminates enzymatic activity; List Biological Laboratories). ...

Anthrax PA binding assays:

Purified murine or human B cells were cultured at 4°C for 30 min with FITC-labeled anthrax PA (50 μg/ml; List Biological Laboratories) in the presence or absence of unlabeled anthrax PA (150 μg/ml) to confirm specific binding. Stained cells were then washed with PBS and analyzed by flow cytometry (see below). Unstained cells were analyzed in parallel to establish background levels of autofluorescence.

ELISA:

Primary B cells were cultured in complete RPMI for 4 to 5 h, washed with RPMI 1640, and then stimulated as indicated in the presence or the absence of anthrax LT for 7 days.  ...

Murine in vivo studies:

Mice were treated with varying doses of anthrax LT as indicated, using a fixed ratio of LF/PA of 1:2.5. LF and PA were resuspended in PBS and injected i.p. into mice in a total volume of 1.0 ml of PBS. As a negative control, selected mice were treated with PBS alone. Mice were sacrificed 3 h after treatment, and spleens were harvested for primary B cell isolation as previously described. Primary B cells were then evaluated for proliferation and IgM production.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

PubMed ID: 16670324