Green fluorescent protein as a signal for protein-protein interactions.

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances

Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein optimised for yeast (yEGFP3) under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances. The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances. The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption. Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances. The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay.     

High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the GreenScreen HC GADD45a-GFP genotoxicity assay

The battery of genetic toxicity tests required by most regulatory authorities includes both bacterial and mammalian cell assays and identifies practically all genotoxic carcinogens. However, the relatively high specificity of the Salmonella mutagenicity assay (Ames test) is offset by the low specificity of the established mammalian cell assays, which leads to difficulties in the interpretation of the biological relevance of results. This paper describes a new high-throughput assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP). A study of 75 well-characterised genotoxic and non-genotoxic compounds with diverse mechanisms of DNA-damage induction (including aneugens) reveals that the assay responds positively to all classes of genotoxic damage with both high specificity and high sensitivity. The current micro-well assay format does not include metabolic activation, but a separate low-throughput protocol demonstrates a successful proof-of-principle for an S9 metabolic activation assay with the model pro-mutagen cyclophosphamide. The test should be of value both as a tool in the selection of candidate compounds for further development, where additional data may be required because of conflicting information from the in vitro test battery, or in product development areas where the use of animals is to be discontinued. As a microplate assay however, it has the qualities of high throughput and low compound use that will facilitate its application in early screening for genotoxic liability.     

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently‐labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP‐expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single‐band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Histological analysis of GFP expression in murine bone.

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a beta-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.     

A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.     

Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.     

Use of the SOS-chromotest spot assay as a screening system for detecting genotoxic compounds in crude plant extracts

An SOS-chromotest spot assay was used to detect genotoxic compounds in crude plant extracts. The method allows simultaneous testing of extracts from different species in either a liquid or a solid crystalline form. Extracts from two species of the genus Senna, native to the state of Morelos, Mexico, were assayed. Four genotoxic compounds were isolated, and were identified as quercetin and rutin from S. wislizeni, and 5,7-di- O-methylrutin and 5,7-di-O-methylquercetin from S. skinneri. The SOS-chromotest spot assay proved to be useful for activity- guided fractionation at the beginning of screening for genotoxic compounds in crude plant extracts.     

Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies

We demonstrate a novel bio‐spectroscopic technique, “simultaneous Raman/GFP microspectroscopy”. It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti‐Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two‐component multivariate curve resolution with alternate least squares (MCR‐ALS) analysis in the anti‐Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR‐ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm^?1, previously reported as “Raman spectroscopic signature of life of yeast.”     

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)–fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134–206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V_max and k_cat) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.     

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999     

A prototype microfluidic chip using fluorescent yeast for detection of toxic compounds

A microfluidic chip has been developed to enable the screening of chemicals for environmental toxicity. The microfluidic approach offers several advantages over macro-scale systems for toxicity screening, including low cost and flexibility in design. This design flexibility means the chips can be produced with multiple channels or chambers which can be used to screen for different toxic compounds, or the same toxicant at different concentrations. Saccharomyces cerevisiae containing fluorescent markers are ideal candidates for the microfluidic screening system as fluorescence is emitted without the need of additional reagents. Microfluidic chips containing eight multi-parallel channels have been developed to retain yeast within the chip and allow exposure of them to toxic compounds. The recombinant yeast used was GreenScreentrade mark which expresses green fluorescent proteins when is exposed to genotoxins. After exposure of the yeast to target compounds, the fluorescence emission was detected using an inverted microscope. Qualitative and quantitative comparisons of the fluorescent emission were performed. Results indicated that fluorescent intensity per area significantly increases upon exposure to methyl-methanesulfonate, a well known genotoxic compound. The microfluidic approach reported here is an excellent tool for cell-based screening and detection of different toxicities. The device has the potential for use by industrial manufacturers to detect and reduce the production and discharge of toxic compounds, as well as to characterise already polluted environments.     

A multiwavelength fluorescence probe: is one probe capable for on-line monitoring of recombinant protein production and biomass activity?

Monitoring cell culture performance requires maximizing the number and the quality of measured parameters and in situ 2D fluorescence spectroscopy could allow intensification of simultaneous data acquisition. The use of a multiwavelength fluorescence probe is proposed for monitoring GFP-producing cultures in bioreactor. The yeast Pichia pastoris and NSO mammalian cells were studied as model systems. Tryptophan, NAD(P)H and riboflavins (riboflavin, FMN, FAD) signals were effective for on-line yeast biomass estimation during the growth phase. During the GFP production phase, in situ measurements of the GFP concentration from the fluorescence probe were well correlated with off-line analyses. Tryptophan and NAD(P)H signals diverged from that of biomass during GFP production. With NSO mammalian cells, results showed that the culture parameters have to be optimized for the use of a fluorescence probe. The use of serum and phenol-red interfered with NAD(P)H and riboflavins fluorescence signals. Nevertheless, it appears that a multiwavelength probe could be useful for culture monitoring of biomass, cell activity and recombinant protein expression in an optimized culture medium.     

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full‐length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full‐length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark‐derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A‐GFP system. Yeast library screening of full‐length vNAR variants which were detected via GFP expression yielded the same high‐affinity binder that had previously been isolated by our group using the conventional epitope tag‐based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost‐friendly alternative to conventional epitope tag detections.     

Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18

The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.     

Two-photon excitation in fluorescence polarization receptor-ligand binding assay

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.     

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.     

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z'' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.