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Sylvia Daunert – One of the best experts on this subject based on the ideXlab platform.

  • red shifted Aequorin variants incorporating non canonical amino acids applications in in vivo imaging
    PLOS ONE, 2016
    Co-Authors: Kristen Marie Grinstead, Laura Rowe, Emre Dikici, Charles Mark Ensor, Smita Joel, Pirouz Daftarian, Jean Marc Zingg, Sylvia Daunert

    Abstract:

    The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of Aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by Aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows Aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, Aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the Aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that Aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

  • Aequorin mutants with increased thermostability
    Analytical and Bioanalytical Chemistry, 2014
    Co-Authors: Xiaoge Qu, Mark Ensor, Laura Rowe, Emre Dikici, Sylvia Daunert

    Abstract:

    Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein Aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double Aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type Aequorin during thermostability studies at 37 °C. Moreover, the quadruple Aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable Aequorin mutant identified thus far.

  • Aequorin variants with improved bioluminescence properties.
    Protein engineering design & selection : PEDS, 2009
    Co-Authors: Emre Dikici, Mark Ensor, Laura Rowe, Sapna K. Deo, L. Millner, C. Logue, Sylvia Daunert

    Abstract:

    The photoprotein Aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoAequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of Aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic Aequorins. Variants of Aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two Aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Sapna K. Deo – One of the best experts on this subject based on the ideXlab platform.

  • Aequorin variants with improved bioluminescence properties.
    Protein engineering design & selection : PEDS, 2009
    Co-Authors: Emre Dikici, Mark Ensor, Laura Rowe, Sapna K. Deo, L. Millner, C. Logue, Sylvia Daunert

    Abstract:

    The photoprotein Aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoAequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of Aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic Aequorins. Variants of Aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two Aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

  • Handbook of Biosensors and Biochips – Recombinant Aequorin‐Based Systems for Biomarker Analysis
    Handbook of Biosensors and Biochips, 2008
    Co-Authors: Laura Rowe, Mark Ensor, Emre Dikici, Sapna K. Deo, Krystal Teasley, Sylvia Daunert

    Abstract:

    Detection methods utilized in the development of biosensors and biochips often require labeling strategies. Commonly employed labels include radioisotopes, enzymes, and fluorohphores. Bioluminescent proteins offer an alternative labeling option and are becoming increasingly popular because of their versatility, sensitivity, and nontoxicity. Bioluminescent labels emit light of varying colors as a method of releasing the energy that is produced by their specific chemical reactions. Aequorin is an example of one such bioluminescent protein which has been studied extensively and used in a variety of bioanalytical techniques. In this chapter, we describe the qualities of Aequorin and the potential that it has as a generic label. This chapter also details several Aequorin-based immunoassays that have been developed for the monitoring of biomarkers important to both hormonal disorders and cardiovascular disease. Lastly, we discuss the progress and the pitfalls associated with developing multianalyte detection schemes and robust sensing technologies based on bioluminescent, Aequorin labeling.

    Keywords:

    Aequorin;
    label;
    reporter;
    calcium detection;
    bioluminescence;
    photoprotein;
    immunoassay;
    lab-on-a-CD;
    multianalyte detection

  • Bioluminescence resonance energy transfer from Aequorin to a fluorophore: an artificial jellyfish for applications in multianalyte detection
    Analytical and Bioanalytical Chemistry, 2005
    Co-Authors: Sapna K. Deo, Mara Mirasoli, Sylvia Daunert

    Abstract:

    In nature, the green light emission observed in the jellyfish Aequorea victoria is a result of a non-radiative energy transfer from the excited-state Aequorin to the green fluorescent protein. In this work, we have modified the photoprotein Aequorin by attaching selected fluorophores at a unique site on the protein. This will allow for in vitro transfer of bioluminescent energy from Aequorin to the fluorophore thus creating an “artificial jellyfish”. The fluorophores are selected such that the excitation spectrum of the fluorophore overlaps with the emission spectrum of Aequorin. By modifying Aequorin with different fluorophores, bioluminescent labels with different emission maxima are produced, which will allow for the simultaneous detection of multiple analytes. By examining the X-ray crystal structure of the protein, four different sites for introduction of the unique cysteine residue were evaluated. Two fluorophores with differing emission maxima were attached individually to the mutants through the sulfhydryl group of the cysteine molecule. Two of the fluorophore-labeled mutants showed a peak corresponding to fluorophore emission thus indicating resonance energy transfer from Aequorin to the fluorophore.

Satoshi Inouye – One of the best experts on this subject based on the ideXlab platform.

  • Slow luminescence kinetics of semi-synthetic Aequorin: expression, purification and structure determination of cf3-Aequorin
    Journal of biochemistry, 2018
    Co-Authors: Satoshi Inouye, Yuri Tomabechi, Takamitsu Hosoya, Shun-ichi Sekine, Mikako Shirouzu

    Abstract:

    cf3-Aequorin is one of the semi-synthetic Aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native Aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3-CTZ) and the histidine-tagged apoAequorin expressed in Escherichia coli cells. The purified cf3-Aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native Aequorin, and its luminescence capacity was estimated to be 72.8% of that of native Aequorin. The crystal structure of cf3-Aequorin was determined at 2.15 A resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p-hydroxy moiety at the C6-phenyl group. In native Aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3-Aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3-CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3-Aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3-Aequorin.

  • Purification of histidine-tagged Aequorin with a reactive cysteine residue for chemical conjugations and its application for bioluminescent sandwich immunoassays
    Protein Expression and Purification, 2012
    Co-Authors: Satoshi Inouye, Jun'ichi Sato

    Abstract:

    Abstract Highly purified histidine-tagged Aequorin with a reactive cysteine residue (His-Cys4-Aequorin) was obtained from the periplasmic space of Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3 mg of His-Cys4-Aequorin from 2 L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-Aequorin with maleimide-acitivated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for α-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01–100 ng/ml and the sensitivities were similar to that using Aequorin-labeled specific antibody and amino-biotinylated Aequorin.

  • Recombinant Aequorin with a reactive cysteine residue for conjugation with maleimide-activated antibody
    Analytical biochemistry, 2008
    Co-Authors: Satoshi Inouye, Jun'ichi Sato

    Abstract:

    Abstract The mutated recombinant Aequorin with a reactive cysteine residue (Cys–Aequorin) was highly purified and then conjugated with a maleimide-activated antibody without significant loss of luminescence activity. The conjugate ratio of Cys–Aequorin to heavy chain of immunoglobulin G (IgG) was estimated to be 1:1. To test the bioluminescent immunoassay with Aequorin-labeled antibody, α-fetoprotein (AFP), a serological marker of liver cancer, was used as a model analyte. The measurable range of AFP was 0.02 to 200 ng/ml with the coefficient of variation between 2.1 and 4.5%.