Contents:

Chapter 1 Executive Summary Purpose and Scope Summary Additional Information Chapter 2 Introduction Historical Perspective Bioluminescence, pre-1960 Aequorea Victoria, GFP, and the 1960s GFP Characterizations from 1970 to 1992 GFP Fundamentals GFP Characterizations GFP Evaluations Cell Biology Applications Drug Discovery Applications Definitions Select Luminescence Terms Select Fluorescence Terms Select Bioluminescence Terms Chapter 3 GFP Fundamentals General Biology of Aequorea Victoria and GFP Phyletic Distribution of GFP and “GFP-like” Proteins Hydrozoans Anthozoans Function of GFP in Aequorea Victoria Geographical Distribution Life Cycle Habitat and Environment Anatomic Localization of GFP in Aequorea Victoria Nomenclature Molecular Biology of GFP Recombinant Aequorea GFP (wt) Chromophore GFP Protein Structure Chemical Reagents and Factors That Affect GFP Stability Factors That Can Perturb GFP Fluorescence pH Effects and GFP Dimerization Denaturation Some “Windows” Within Which GFP is Stable Organic Solvents and GFP Proteases and GFP Detergents and GFP Oxidizing and Reducing Agents and GFP Chaotropes Physical Characteristics of GFP Fluorescence Fluorescence Measurements Excitation Emission Molar Extinction Coefficients Quantum Yield Stokes Shift Fluorescence Lifetime Energy Transfer Radiative (Trivial) Energy Transfer Radiationless Energy Transfer Common Problems in GFP Fluorescence Detection Autofluorescence Recognizing Autofluorescence Reducing Autofluorescence Photostability Photobleaching Photoisomerization Photoconversion Chapter 4 GFP Variant Evaluation Parameters to Consider when Evaluating GFP Variants Brightness Autofluorescence Photostability Solubility, Thermostability, and Folding Stokes Shift Environmental Stability Codon Bias Maturation Times Turnover Rate Monomeric Forms Cryptic Introns Characterized GFP and “GFP-like” Variants Color Palette of GFP and “GFP-like” Variants Blue-Fluorescent Proteins (BFPs) Cyan-Fluorescent Proteins (CFPs) Green-Fluorescent Proteins (GFPs) Yellow-Fluorescent Proteins (YFPs) Red-Fluorescent Proteins (RFPs) Far-Red Fluorescent Proteins Green-Emitting Fluorescent Proteins from Corals Selected Variants Classed by Phenotypes other than Color Destabilized GFP Variants Photoactivation and GFP Variants Available Crystallography Data Sample Checklist of Questions for GFP Variant Evaluation Chapter 5 GFP Gene Cloning and Expression Commercially Available GFP Cloning Vectors Vectors for Specific Expression Systems Vectors for Specific Applications Arranging and Re-arranging GFP Clone Architecture Promoters Kozak Sequences Targeting to Subcellular Compartments Affinity and Epitope Tags Cloning Checkpoints GFP Fusion Proteins Amino or Carboxy Fusions Linkers GFP Fusion Partners GFP Fusion to a Wide Range of Proteins Bioluminescence Resonance Energy Transfer (BRET) GFP Gene Delivery Transient Gene Transfer Stable Transformants GFP Licenses GFP Gene Expression Cell Culture Expression Systems Bacterial Expression Systems Mammalian Expression Systems Insect Expression Systems Whole Organism Model Systems Vertebrate Model Systems Zebrafish Xenopus Invertebrate Model Systems Caenorhabditis Elegans Drosophila Melanogaster Chapter 6 GFP Protein Purification Traditional Preparative Purification Methods Common Pre-column Steps Cell Disruption Clarification Ammonium Sulfate Precipitation Common Chromatography Methods Ion Exchange Chromatography Affinity Purification Hydrophobic Interaction Chromatography Gel Filtration and Size Exclusion Chromatography Organic Extraction of GFP Analytical Protein Purification High-Throughput Protein Purification Chapter 7 Fluorescence Detection and GFP General Considerations Available Light Sources for GFP Excitation GFP Emission Spectra and Fluorescence Detection Systems The Role of Filter Selection Instrument Sensitivity Spectrophotometry Fluorimetry Fluorometric Plate Readers Flow Cytometry Native PAGE Electrophoresis Fluorescence Microscopy and Imaging Environment Sample Preparation and Maintenance Fluorescence Microscope Set-Up Types of Fluorescence Microscopy Wide-Field Fluorescence Microscopy Confocal Microscopy Two Photon Excitation Microscopy (TPEM) Advanced Fluorescence Microscopy Techniques Fluorescence Recovery After Photobleaching (FRAP) Fluorescence Loss in Photobleaching (FLIP) Fluorescence Correlation Microscopy (FCM) Fluorescence Lifetime Imaging (FLIM) Fluorescence Resonance Energy Transfer (FRET) Microscopy Total Internal Reflection Fluorescence Microscopy (TIR-FM) Chapter 8 Calibrations and Data Analysis Data Output from Spectrophotometers A280 Readings Wavelength Scans Molar Extinction Coefficients (MEC’s) Data Output from Fluorimeters Data Output from Fluorometric Plate Readers Data Output from Flow Cytometry Data Output from Native PAGE Electrophoresis Data Output from Microscopy and Imaging Cameras Image Capture Image Analysis Image Storage Chapter 9 GFP Applications In Cell Biology GFP at Various Levels of Cell Biology GFP at the Cellular Level GFP at the Subcellular Level GFP and Organelle Behavior GFP and Protein Behavior GFP and Ion Behavior Strategies for Monitoring GFP in Cell Biology Living or Fixed Cells Single Color or Multi-Color Spatial or Temporal Qualitative or Quantitative Advantages of Using GFP in Cell Biology Limitations of Using GFP in Cell Biology Experimental Considerations Chapter 10 GFP Applications In Drug Discovery Assay Detection Modes and Drug Discovery Analysis of Various Detection Modes Fluorescence Alternative Modes of Assay Detection Relevant GFP Characteristics Advantages of GFP Relevant to Drug Discovery Limitations of GFP in Drug Discovery Drug Discovery Process Specific Applications of GFP in Drug Discovery Target Identification and Validation GFP-Based Assays and Target Biology GFP and Protein (Target) Families Lead Identification Cellular Assays High-Content Screening (HCS) Drug Development GFP in Toxicology Whole Organism Assays Chapter 11 GFP as an Educational Tool in Biotechnology GFP as an Educational Tool Workshops Biotechnology Educational Kits Chapter 12 Emerging Technologies New GFP Variants Developments in Model Systems Developments in Detection Instruments Array Technology Developments in Drug Discovery The Future of GFP Chapter 13 Appendices Vendors GFP-Related Books Relevant Journals Societies Symposia Educational Workshops Online Protocols GFP-Related Web Sites Selected Media Reports GFP Fact Sheet Chapter 14 References

Summary:

Purpose and Scope The purpose of this Guide is to assist academic, biotechnology, and pharmaceutical professionals in their evaluation of the utility of green-fluorescent protein (GFP) and its variants. In parallel, it is hoped that this Guide will serve as useful introductory text and practical resource for the incorporation of GFP into various research and drug discovery programs. The Guide presents an overview of fundamental facts that relate to GFP and its variants. The reader will be taken through aspects of GFP variant selection criteria, examples of available GFP vectors, expression systems, and suitable protein purification techniques. Examples of appropriate fluorescence detection instrumentation are provided, in addition to some illustrations of how GFP is currently being used in cell biology and drug discovery. Strengths and weakness of GFP variants and associated technology are highlighted, where appropriate. In the past decade, GFP has revolutionized cell biology. Since its cloning in 1992, GFP has become one of the most widely used indicators of gene induction in living cells. As a genetically encoded protein, GFP is used to track protein movement in cells, and to monitor cellular events from cell division to cancer metastasis. This has had far-reaching implications in basic sciences such as cellular and developmental biology, neurobiology, and molecular biology and applied sciences including drug discovery. The Guide will illustrate how GFP is beginning to emerge as a valuable tool in drug screening and development. This Guide is not a handbook of laboratory protocols but a guide that can exist alongside such a handbook in providing resources, suggestions, pitfalls, and some practical “know how” in GFP applications. Knowing some of the strengths and limitations of GFP, its variants, and associated technology should facilitate the reader’s getting the most from GFP, enabling the protein to live up to its reputation as an outstanding reporter of cellular processes. Summary The green-fluorescent protein (GFP), a protein that emits vivid green light upon excitation has, in the past decade, secured a place as one of the most remarkable tools in modern day biology. As GFP confers its color to a particular cellular structure or component, visualization of “live” cellular events has become possible and has brought rapid progress in cell biology. Disease areas that have benefited from these advances include neurology, oncology, endocrinology, cardiology, and diabetes to name a few. GFP biology has known many significant milestones in the past 40 years but four of these deserve special mention. First, in 1962 it was realized that the green “glow” from biochemical isolates of the luminescent Pacific Northwest jellyfish, Aequorea victoria, was protein-based. Second, in 1992, A.victoria GFP was cloned; and third, in 1994, GFP was expressed for the first time in several expression model systems requiring no co-factors to create the vivid fluorescence. Fourth, in 1999, cloning of fluorescent proteins from non-luminous Indo-Pacific reef corals was reported. These latter proteins bear similarities to GFP, they have become utilized in parallel to GFP, and they are informally categorized as “GFP-like” proteins. As a result of these and many other key findings, the past decade has seen increases in GFP-related publications from singledigit numbers in 1992 to more than 10,000 in 2003. These citation numbers are testimony to the versatility and applicability of this exceptional “super-family” of fluorescent proteins, now numbering 30 or upwards in mainstream cloned varieties. Despite its remarkable usefulness, limitations of Aequorea GFP (wt) exhibits some limitations. Reported limitations include problems with expression level, photosensitivity, low brightness, and insolubility. These limitations were a driving force for efforts to improve GFP by random mutagenesis of the gene. These genetic manipulations generated variants with new colors (blue, cyan, and yellow) and those that are more soluble, photostable, and thermostable. Since elucidation of the 3D crystallographic structure of GFP (and its variants), random mutagenesis has been replaced, in part, by site-directed mutagenesis. Some GFPs or GFP-like variants, while bringing new or improved features, have other unexpected problems. These include slow maturation, tetramerization, and the potential for dimerization, aggregation, or mis-targeting. A new fluorescent protein cDNA clone is less likely, therefore, to have all the kinks removed and is likely to need several rounds of mutagenesis before becoming optimized for use in cell-based applications. One attractive feature of GFP is its unusual stability. However, even in this markedly stable protein, there are conditions that can cause GFP fluorescence to be diminished. Knowing or finding out which experimental factors may reduce or perturb GFP fluorescence is an important part of the search for, and optimization of, GFP applications. Factors such as pH extremes, extremes of heat, high concentrations of organic solvents, compromised photostability, or autofluorescence can diminish the usefulness of GFP as a reporter. Among the GFPs in common use, recombinant Aequorea GFP (wt) has been characterized most fully with respect to its response to common detergents, chaotropes, proteases, reducing agents, and some oxidizing agents. Practical applications of GFP have been advanced by understanding its 3D structure. The architecture of this barrel-like structure provides insight into GFP resistance to major moleculardisruption and major deletions. The positions of the amino and carboxy termini on the same end of a compact barrel structure afford some understanding about the success of many GFP gene fusion experiments. In practical terms, producing parallel fusions, one at the amino terminus of GFP, the other at its carboxy terminus may improve the chances of success as not all fusions are guaranteed to succeed. Fusion partner biological activity should be tested as early as possible. Perturbations of GFP, its fusion partner, or the cell are expected to be minimal, but experiments should be undertaken to determine the extent of such perturbations. GFP has been expressed in many model systems used in cell, molecular, and developmental biology. Model systems utilized are mainly cells in culture (e.g., E.coli, insect, or mammalian cells) or whole organisms (e.g., zebrafish, Xenopus, Drosophila). Often new GFP variants are designed for expression within a particular phylogenetic group of organisms that share common codon bias. Such genetic manipulations may be “silent” with respect to amino acid sequence and protein structure, but may result in improved expression levels. Expressed in whole organisms, GFP is proving to be a useful, non-invasive detection tool. One example is the use of GFP in oncology, as a probe for detecting metastasis. Sometimes, large amounts of pure GFP must be obtained. This is the case when raising antibodies to GFP, when using GFP as a protein standard, or when testing the in vitro activity of GFP fusion proteins. For such purposes, GFP must be purified to near homogeneity. Although GFP can be purified by traditional protein purification methods, a popular and more rapid choice is affinity chromatography. Other choices include various organic extraction methods such as ones that combine aqueous salt solutions with polar organic solvents. The stability and easy tracking of GFP make it a particularly useful demonstration protein. Whole courses and modular curricula in protein chemistry and molecular biology have been designed around Aequorea GFP. GFP can also act like an epitope tag, a useful characteristic when a fusion partner does not have a specific antibody. The purification of large numbers of GFP fusion proteins is now possible, asmicroplate platforms have been engineered to bind GFP specifically. For decades fluorescence monitoring has been used in diagnostics and drug testing. High sensitivity, ease of detection, and excellent dynamic range have contributed to the success of fluorimetry. So, when GFP emerged on the scene in 1994, a wide range of proven fluorimetric methods were at hand. GFP fluorescence can be detected, for example, by spectrofluorimetry, flow cytometry, fluorimetric plate reading, fluorescence microscopy, and fluorimetric imaging. Choosing appropriate instrumentation and optimizing optical parameters are always challenges. The careful selection of lasers and filters and the understanding of salient features of excitation and emission spectra are important requirements. Such knowledge can maximize signal while suppressing background noise and autofluorescence. Additional challenges in microscopy and imaging are data/image analysis and storage. These should be considered at the outset of an experimental program. Capabilities of GFP have been demonstrated in two, three, or four dimensions—the fourth dimension, temporal dynamics of protein and organelle migration, being captured by time-lapse photography or video imaging. Such data can be multiplexed with two or more color variants of GFP, each of which reports on a different event. However, with more complex multi-channel monitoring comes the need to minimize spectral overlap. Quantitative microscopy is also becoming possible, as methods for internal and external GFP calibration become available. GFP’s already-proven applications in cell biology have paved the way for cell-based drug discovery applications. GFP gene fusion technology has facilitated spatial and temporal analysis of GFP-labeled cell structures, organelles, and proteins in living cells. In drug discovery, the ability to fuse gfp with genetically encoded drug targets allows GFP to act as an embedded reporter of target behavior in cell-based drug screens. This approach is in alignment with current trends in the drug discovery industry in which data-rich, cell-based assays are highly desirable.Other cell-based applications integrated GFP into protein localization assays and protein folding assays. Developments in high-throughput, live-cell imaging via high-content screening expands the capability of cell-based assays (including those that utilize GFP). The end point for medical research is not only understanding the “whys and wherefores” of a particular disease but providing remedies to prevent or minimize disease pathology. Perhaps the most amazing facets of GFP (and other fluorescent proteins) have been their ability to literally shed light on disease at the cellular and subcellular level. The challenge now is to establish the potential utility of GFP in drug discovery technology. From “jellyfish to clinic” could be a fairly interesting, albeit, circuitous route. In reality, this could demonstrate the ultimate realization of GFP potential. Perhaps the jellyfish is not so humble. Additional Information This Guide is not an exhaustive or in-depth review about GFP (or its variants). Instead, presented is an introductory, resource-orientated overview of GFP with particular emphasis on practical applications. For further information, readers are directed (in the Guide) to salient texts, reviews, or specific articles on GFP. As an additional guide to GFP-related information, the Appendices lists Web sites, societies, events, conferences, and relevant workshops. Suppliers of major fluorescence detection instrumentation and associated technology in areas such as confocal microscopy, imaging/image analysis, and flow cytometry provide high-quality support ranging from Web site information to customer support and training courses. Their specific Web sites may be consulted for further information. Some relevant training courses are listed in the Appendix. Many vendors and suppliers of components of GFP technology are also mentioned in the text and are listed in the Appendix. These listings include instrument specialists, molecular tool suppliers, drug discovery tool suppliers, suppliers of assays already incorporating GFP, and suppliers of GFP clones and antibodies. Companies or vendors are listed as examples, but their mention does not serve as endorsement.