Photoproteins in Bioanalysis

The use of light–emitting proteins for the detection of biomolecules provides fast and sensitive methods which overcome the disadvantages of radioactive labels and the high cost of fluorescent dyes.
This reference work summarizes modern advanced techniques and their applications and includes practical examples of assays based on photoproteins. The book presents contemporary key topics like luminescent marine organisms, DNA probes, reporter gene assays and photoproteins, ratiometric sensing, use of photoproteins for in vivo functional imaging and luminescent proteins in binding assays, to name just a few, and is complemented by recent advances in instrumentation.

Spis treści:
Preface.
List of Contributors.
1 The Photoproteins (Osamu Shimomura).
1.1 Discovery of Photoprotein.
1.2 Various Types of Photoproteins Presently Known.
1.2.1 Radiolarian (Protozoa) Photoproteins.
1.2.2 Coelenterate Photoproteins.
1.2.3 Ctenophore Photoproteins.
1.2.4 Pholasin (Pholas Luciferin).
1.2.5 Chaetopterus Photoprotein.
1.2.6 Polynoidin.
1.2.7 Symplectin.
1.2.8 Luminodesmus Photoprotein.
1.2.9 Ophiopsila Photoprotein.
1.3 Basic Strategy of Extracting and Purifying Photoproteins.
1.4 The Photoprotein Aequorin.
1.4.1 Extraction and Purifi cation of Aequorin.
1.4.1.1 Hydrophobic Interaction Chromatography.
1.4.2 Properties of Aequorin.
1.4.2.1 Stability.
1.4.2.2 Freeze–drying.
1.4.3 Specifi city to Ca<sup>2+</sup>.
1.4.4 Luminescence of Aequorin by Substances Other Than Divalent Cations.
1.4.5 Mechanism of Aequorin Luminescence and Regeneration of Aequorin.
1.4.5.1 Structure of Aequorin.
1.4.5.2 Luminescence Reaction.
1.4.5.3 Regeneration.
1.4.6 Inhibitors of Aequorin Luminescence.

1.4.7 Recombinant Aequorin.
1.4.8 Semi–synthetic Aequorins.
1.4.8.1 e–A equorins.
References.
2 Luminous Marine Organisms (Steven H.D. Haddock).
2.1 Introduction.
2.1.1 Non–luminous Taxa.
2.1.2 Luminous Taxa.
2.2 Taxonomic Distribution of Bioluminescence.
2.2.1 Bacterial Luminescence.
2.2.2 Dinofl agellate Luciferin.
2.2.3 Cypridina (Vargula) Luciferin.
2.2.4 Coelenterazine.
2.2.5 Other Luciferins: Known and Unknown.
2.3 Functions.
2.3.1 Startle or Distract.
2.3.2 Burglar Alarm.
2.3.3 Counterillumination.
2.3.4 Mating Displays.
2.3.5 Prey Attraction.
References.
3 Beetle Luciferases: Colorful Lights on Biological Processes and Diseases (Vadim R. Viviani and Yoshihiro Ohmiya).
3.1 Introduction.
3.2 Beetle Luciferases.
3.3 Bioanalytical Assays of ATP.
3.3.1 Biomass Estimation and Microbiological Contamination.
3.3.2 Cytotoxicity and Cell Viability Tests.
3.3.3 Enzymatic Assays.
3.4 Luciferases as Reporter Genes.
3.4.1 Dual and Multiple Reporter Assays.
3.5 Biophotonic Imaging in Animals: A Living Light on Diseases.
3.5.1 Pathogen Infection in Living Models.
3.5.2 Drug Screening.
3.5.3 Tumor Proliferation and Regression Studies.
3.5.4 Gene Delivery and Gene Therapy.
3.5.5 Luciferase as Biomarkers for Cell Traffi cking Studies.
3.5.6 Immunoassays.
3.6 Biophotonic Imaging in Plants.
3.7 Biosensors: Sensing the Environment.
3.8 Novel Luciferases: Different Colors for Different Occasions.
References.
4 Split Luciferase Systems for Detecting Protein–Protein Interactions in Mammalian Cells Based on Protein Splicing and Protein Complementation (Yoshio Umezawa).
4.1 Introduction.
4.2 Protein Splicing–based Split Firefl y Luciferase System [23].
4.2.1 Split Luciferase Works as a Probe for Protein Interaction.
4.3 Split Renilla Luciferase Complementation System [33].
4.3.1 Time Course of the Interaction Between Y941 and SH2n.
4.3.2 Location of the Interaction Between Y941 and SH2n.
References.
5 Photoproteins in Nucleic Acid Analysis (Theodore K. Christopoulos, Penelope C. Ioannou, and Monique Verhaegen).
5.1 Hybridization Assays.
5.2 Quantitative Polymerase Chain Reaction.
5.3 Genotyping of Single–nucleotide Polymorphisms.
5.4 Conjugation of Aequorin to Oligodeoxynucleotide Probes.
5.5 Development of New Recombinant Bioluminescent Reporters.
5.6 Signal Amplifi cation by in Vitro Expression of DNA Reporters Encoding Bioluminescent Proteins.
5.7 Conclusions.
References.
6 Bioluminescence Resonance Energy Transfer in Bioanalysis (Suresh Shrestha and Sapna K. Deo).
6.1 Introduction.
6.2 BRET Principle, Effi ciency, and Instrumentation.
6.3 Comparison of BRET and FRET.
6.4 Examples of BRET Donor–Acceptor Pairs.
6.5 Applications of BRET in Bioanalysis.
6.5.1 Homogeneous Assays.
6.5.2 Protein–Protein Interactions and High–throughput Screening.
6.6 Conclusions.
References.
7 Photoproteins as in Vivo Indicators of Biological Function (Rajesh Shinde, Hui Zhao, and Christopher H. Contag).
7.1 Overview.
7.2 Probes Used for in Vivo Bioluminescence Imaging.
7.3 Probes Used for in Vivo Fluorescence Imaging.
7.4 Detection Technologies.
7.5 Current Applications.
7.5.1 Oncology.
7.5.2 Infectious Disease.
7.5.3 Bacterial Infections.
7.5.4 Viral Infections.
7.5.5 Viral–mediated Gene Transfer.
7.5.6 Cell Biology.
7.5.7 Stem Cell Biology.
7.6 Protease Sensors.
7.7 Conclusions.
References.
8 Photoproteins as Reporters in Whole–cell Sensing (Jessika Feliciano, Patrizia Pasini, Sapna K. Deo, and Sylvia Daunert).
8.1 Introduction.
8.1.1 Biosensors Using Intact Cells.
8.1.2 Reporter Genes in Genetically Engineered Whole–cell Sensors.
8.2 The L uciferases.
8.2.1 Bacterial Luciferases.
8.2.1.1 luxAB Bioreporters.
8.2.1.2 luxCDABE Bioreporters.
8.2.1.3 Naturally Luminescent Bioreporters.
8.2.2 Eukaryotic Luciferases.
8.2.2.1 Firefl y Luciferase.
8.2.2.2 Sea Pansy Luciferase.
8.3 Aequorin.
8.4 Fluorescent Proteins.
8.4.1 Green Fluorescent Protein.
8.4.2 Red Fluorescent Protein.
8.5 Multiplexing.
8.6 Applications.
8.6.1 Stress Factors and Genotoxicants.
8.6.2 Environmental Pollutants.
8.6.3 Quorum–sensing Signaling Molecules.
8.6.4 Antibiotics.
8.7 Technological Advances.
References.
9 Luminescent Proteins in Binding Assays (Aldo Roda, Massimo Guardigli, Elisa Michelini, Mara Mirasoli, and Patrizia Pasini).
9.1 Introduction.
9.2 Protein–Protein and Protein–Ligand Interaction Assays.
9.2.1 FRET and BRET Techniques.
9.2.2 FRET and BRET Applications.
9.2.3 Other Detection Principles.
9.3 Antibody–based Binding Assays.
9.3.1 Chemical Conjugation.
9.3.2 Gene Fusion.
9.3.3 Dual–analyte Assays.
9.3.4 Expression Immunoassays.
9.3.5 BRET–based Immunoassays.
9.4 Biotin–Avidin Binding Assays.
9.5 Nucleic Acid Hybridization Assays.
9.6 Other Binding Assays.
9.7 Concluding Remarks.
References.
10 Luminescent Proteins: Applications in Microfl uidics and Miniaturized Analytical Systems (Emre Dikici, Laura Rowe, Elizabeth A. Moschou, Anna Rothert, Sapna K. Deo, and Sylvia Daunert).
10.1 Miniaturization and Microfl uidics.
10.2 Photoproteins and Applications in Miniaturized Detection Systems.
10.2.1 Green Fluorescent Protein.
10.2.1.1 GFP in Miniaturized Microfl uidic–based Assays.
10.2.2 Luciferase.
10.2.2.1 Luciferase in Miniaturized Microfl uidic–based Assays.
10.2.3 Aequorin.
10.2.3.1 Aequorin in Miniaturized Microfl uidic–based Assays.
10.3 Future Perspectives.
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