Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Saunders, Matthew; Kim, Heung-Bok; Woods, Travis A.; Nolan, John P.; Sklar, Larry A.; Edwards, Bruce S.; Graves, Steven W.

doi:10.1002/cyto.a.20268

Cited by 19 publications

(29 citation statements)

References 40 publications

(75 reference statements)

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“…When protease is added the GST-X-mV fusion protein is processed at the correct cleavage site liberating mVenus (Figure 1c). A related methodology based on direct coupling to beads, but not based on coupling of high affinity capture antibodies to beads, has been utilized to evaluate enzymatic cleavage of the anthrax lethal factor, Factor Xa, and botulinum neurotoxin Type A (22). Our in solution assay relies on the protease first processing the GST-X-mV fusion protein in solution.…”

Section: Resultsmentioning

confidence: 99%

“…The HIV-1 protease substrates developed for use are native Gag domains containing embedded cleavage sites and are expressed as fluorescent fusion proteins. A limitation of past bead-based approaches for biochemical analyses of protease - substrate cleavage was the compression of dynamic range for quantifying substrate cleavage and assay sensitivity to substrate concentrations (22). We have fully optimized signal to noise assessment of substrates for high fidelity and throughput, which allows rapid and precise analysis of enzyme kinetics.…”

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confidence: 99%

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A Cleavage Enzyme-Cytometric Bead Array Provides Biochemical Profiling of Resistance Mutations in HIV-1 Gag and Protease

et al. 2011

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The majority of protease – substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease – substrate cleavage assay, termed the Cleavage Enzyme – Cytometric Bead Array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein and substrate cleavage can be followed through the loss of fluorescence utilizing cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (kcat/KM) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or non-cleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme – substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

A Cleavage Enzyme-Cytometric Bead Array Provides Biochemical Profiling of Resistance Mutations in HIV-1 Gag and Protease

et al. 2011

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“…Both of these approaches are easily monitored through loss-of-fluorescence assays with flow cytometry, 45,46 which provides excellent discrimination between free vs. bound signals at the surface of microspheres. 46 …”

Section: Resultsmentioning

confidence: 99%

A Microsphere-Supported Lipid Bilayer Platform for DNA Reactions on a Fluid Surface

Fabry-Wood¹,

Fetrow²,

Brown³

et al. 2017

ACS Appl. Mater. Interfaces

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We report a versatile microsphere-supported lipid bilayer system that can serve as a general-purpose platform for implementing DNA nanotechnologies on a fluid surface. To demonstrate our platform, we implemented both toehold-mediated strand displacement (TMSD) and DNAzyme reactions, which are typically performed in solution and which are the cornerstone of DNA-based molecular logic and dynamic DNA nanotechnology, on the surface. We functionalized microspheres bearing supported lipid bilayers (µSLBs) with membrane-bound nucleic acid components. Using functionalized µSLBs, we developed TMSD and DNAzyme reactions by optimizing reaction conditions to reduce non-specific interactions between DNA and phospholipids and to enhance bilayer stability. Additionally, the physical and optical properties of the bilayer were tuned via lipid composition and addition of fluorescently tagged lipids to create stable and multiplexable µSLBs that are easily read out by flow cytometry. Multiplexed TMSD reactions on µSLBs enabled the successful operation of a Dengue serotyping assay that correctly identified all sixteen patterns of target sequences to demonstrate detection of DNA strands derived from the sequences of all four Dengue serotypes. The limit of detection for this assay was 3 nM. Furthermore, we demonstrated DNAzyme reactions on a fluid lipid surface, which benefit from free diffusion on the surface. This work provides the basis for expansion of both TMSD and DNAzyme based molecular reactions on supported lipid bilayers for use in molecular logic and DNA nanotechnology. As our system is multiplexable and results in fluid surfaces, it may be of use in compartmentalization and improved kinetics of molecular logic reactions, and as a useful building block in a variety of DNA nanotechnology systems.

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“…We have previously described microsphere based HTS assays to detect inhibitors of proteases, anthrax lethal factor (LF) and Botulinum neurotoxin A light chain (BoNT/ALC)(14,25,26). The assays employ recombinant fusion proteins consisting of a protease substrate sequence (peptide with cleavage site) fused at one end with a biotinylated attachment sequence and at the other with green fluorescent protein (GFP).…”

Section: Resultsmentioning

confidence: 99%

“…Biotinylated GFP protease substrates for LF, BoNTALC and a protease-resistant substrate (pinpointGFP) were prepared and loaded on streptavidin microspheres as previously described (14,25,26). Additions to wells were in sequence as follows: 1 st , 4 μL protease buffer; 2 nd , 2 μl of a mixture of 1.5 μM LF and 5 nM BoNTALC in protease buffer; 3 rd , 100 nL of test compounds (1 mM in DMSO); and 4 th , 4 μL containing 2×10 5 /ml of each set of substrate-bearing microspheres.…”

Section: Methodsmentioning

confidence: 99%

Cluster cytometry for high‐capacity bioanalysis

et al. 2012

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Flow cytometry specializes in high content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology has flow cytometry’s multi-experiment throughput begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high throughput screening (HTS). To extend performance and automation compatibility we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1536-well plate format. To assess performance we used cell and microsphere based HTS assays that had been well characterized in previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 μL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional four-fold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS and other large scale investigations of biological complexity.

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Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Cited by 19 publications

References 40 publications

A Cleavage Enzyme-Cytometric Bead Array Provides Biochemical Profiling of Resistance Mutations in HIV-1 Gag and Protease

A Cleavage Enzyme-Cytometric Bead Array Provides Biochemical Profiling of Resistance Mutations in HIV-1 Gag and Protease

A Microsphere-Supported Lipid Bilayer Platform for DNA Reactions on a Fluid Surface

Cluster cytometry for high‐capacity bioanalysis

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