A bioluminescent assay for the sensitive detection of proteases

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.     

Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays.     

In vitro fluorescent analysis of preprotein import into chloroplasts

Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al., Plant Physiol. Biochem., 46, 541-549 (2008)). Here, we propose another in vitro protein transport system that combines fluorescence techniques. We attempted to use two types of precursors: a green fluorescent protein (GFP)-fused precursor and a fluorescent dye-labeled one. Both were successfully imported into chloroplasts. However, the fluorescent dye-labeled precursor was more advantageous than the GFP-fused precursor in the in vitro system.     

Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.     

Random PCR-based screening for soluble domains using green fluorescent protein

A novel approach to screen soluble protein domains is presented, by combining tagged random primer polymerase chain reaction (PCR) method and protein-folding assay using green fluorescent protein. Tagged random primer PCR method was used to amplify random DNA fragments from a template cDNA. The PCR products were fused to the green fluorescent protein (GFP) gene. Then solubilities of their translation products were rapidly monitored by the fluorescence of transformed Escherichia coli colonies on plates. We succeeded in cloning four soluble domains from Vav protein using this method. The present method is applicable to all proteins when cDNAs are available.     

Proteolytic activity assayed by subcellular localization switching of a substrate

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.     

An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio

Protein-protein interactions (PPIs) play crucial roles in various biological processes. Among biochemical, genetic, and imaging approaches that have been used for the study of PPIs, visualization of PPIs in living cells is the key to understanding their cellular functions. The bimolecular fluorescence complementation (BiFC) assay represents one of these imaging tools for direct visualization of PPIs in living cells. The BiFC assay is based on the structural complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein when they are fused to a pair of interacting proteins. Although over 10 different fluorescent proteins have been used for BiFC assays, the two nonfluorescent fragments from all of these fluorescent proteins can spontaneously self-assemble, which contributes to background fluorescence and decreases the signal-to-noise (S/N) ratio in the BiFC assay. Here we report the identification of a mutation, I152L, that can specifically reduce self-assembly and decrease background fluorescence in a Venus-based BiFC system. This mutation allows a 4-fold increase in the S/N ratio of the BiFC assay in living cells. This improved Venus-based BiFC system will facilitate PPI studies in various biological research fields.     

A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.     

Single-molecule pull-down for studying protein interactions

This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.     

Stable expression of Anthozoa fluorescent proteins in mammalian cells

BACKGROUND: Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. METHODS: The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. RESULTS: Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. CONCLUSION: We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.     

Are the SSB-Interacting Proteins RecO,RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.     

Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.     

Flow cytometric screening of cDNA expression libraries for fluorescent proteins

Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.     

Flow cytometric quantification and characterization of intracellular protein aggregates in yeast

The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.     

Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.     

Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives.

Nuclear receptors represent a very good family of protein targets for the prevention and treatment of diverse diseases. In this study, we screened natural compounds and their derivatives, and discovered ligands for the retinoic acid receptors (RARs) and the farnesoid X receptor (FXR). In the reporter assay systems of nuclear receptors presented here, two fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for detection of a ligand-based induction and as an internal control, respectively. By optimizing the conditions (e.g., of hormone response elements and promoter genes for reporter plasmids), we established a battery of assay systems for ligands of RARs, retinoid X receptor (RXR) and FXR. The screening using the reporter assay system can be carried out without the addition of co-factors or substrates. As a result of screening of more than 140 compounds, several compounds were detected which activate RARs and/or FXR. Caffeic acid phenylethyl ester (CAPE), known as a component of propolis from honeybee hives, and other derivatives of caffeic acid up-regulated the expression of reporter gene for RARs. Grifolin and ginkgolic acids, which are non-steroidal skeleton compounds purified from mushroom or ginkgo leaves, up-regulated the expression of the reporter gene for FXR.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.     

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

BACKGROUND: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. METHODS: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. RESULTS: A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. CONCLUSIONS: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.