Bioluminescence Detection of Proteolytic Bond Cleavage by Using Recombinant Aequorin

Deo, Sapna K.; Lewis, John L.; Daunert, Sylvia

doi:10.1006/abio.2000.4539

Cited by 24 publications

(12 citation statements)

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“…Highly sensitive binding assays and immunoassays have been developed for peptidic and nonpeptidic analytes using aequorin as a label (6, 7). A proteolytic bond cleavage assay has also been developed using an aequorin fusion protein (8). Since aequorin can be detected down to attomole levels, it has been employed in the determination of biotin in single cells and in picoliter-volume vials (9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

Cysteine-Free Mutant of Aequorin as a Photolabel in Immunoassay Development

et al. 2002

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The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.

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Section: Introductionmentioning

confidence: 99%

Cysteine-Free Mutant of Aequorin as a Photolabel in Immunoassay Development

et al. 2002

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“…This photoprotein has been widely employed in the measurement of Ca 2+ in various cellular compartments and to monitor gene expression (6,7). It has also been used as a highly sensitive label in binding assays (8)(9)(10)(11)(12) and in the detection of proteolytic bond cleavage (13). Aequorin can be detected in the femtomole to attomole range, which has allowed its employment as an alternative label to fluorescent probes in the calibration of micropipets (14).…”

Section: Introductionmentioning

confidence: 99%

C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development

2001

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Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.

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“…The extent and the duration of the inhibition are proportional to the antioxidant concentration and activity. The luminol/oxidant/enhancer and the hypoxanthine/xanthine oxidase/Fe 2+ -EDTA/luminol CL systems have been extensively used to measure the antioxidant capacity of biological fluids and for screening purposes, including the determination of the antioxidant activity of bile acids [10] and plant natural compounds [11]. Ogawa et al have described an on-line screening method for antioxidants based on liquid chromatography with CL detection [12].…”

Section: Cell-free Assaysmentioning

confidence: 99%

Bioluminescence and chemiluminescence in drug screening

Roda

Guardigli

Pasini

et al. 2003

Analytical and Bioanalytical Chemistry

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Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number of new pharmacological targets. Bioluminescence and chemiluminescence represent suitable detection techniques for high-throughput screening because they allow rapid and sensitive detection of the analytes and can be applied to small-volume samples. In this paper we report on recent applications of bioluminescence and chemiluminescence in drug screening, both for in vitro and in vivo assays. Particular attention is devoted to the latest and most innovative bioluminescence and chemiluminescence-based technologies for drug screening, such as assays based on genetically modified cells, bioluminescence resonance energy transfer (BRET)-based assays, and in vivo imaging assays using transgenic animals or bioluminescent markers. The possible relevance of bioluminescence and chemiluminescence techniques in the future developments of high-throughput screening technologies is also discussed.

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Bioluminescence Detection of Proteolytic Bond Cleavage by Using Recombinant Aequorin

Cited by 24 publications

References 24 publications

Cysteine-Free Mutant of Aequorin as a Photolabel in Immunoassay Development

Cysteine-Free Mutant of Aequorin as a Photolabel in Immunoassay Development

C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development

Bioluminescence and chemiluminescence in drug screening

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