A Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka

Cre/loxP-dependent expression of fluorescent proteins represents a powerful biological tool for cell lineage, fate-mapping, and genetic analysis. Live tissue imaging has significantly improved with the development of far-red fluorescent proteins, with optimized spectral characteristics for in vivo applications. Here, we report the generation of the first transgenic mouse line expressing the far-red fluorescent protein Katushka, driven by the hybrid CAG promoter upon Cre-mediated recombination. After germ line or tissue-specific Cre-driven reporter activation, Katushka expression is strong and ubiquitous, without toxic effects, allowing fluorescence detection in fresh and fixed samples from all tissues examined. Moreover, fluorescence can be detected by in vivo noninvasive whole-body imaging when Katuhska is expressed exclusively in a specific cell population deep within the animal body such as pancreatic beta cells. Thus, this reporter model enables early, widespread, and sensitive in vivo detection of Cre activity and should provide a versatile tool for a wide spectrum of fluorescence and live-imaging applications.     

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.     

Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM beta-lactamase as a reporter in mammalian cells. The beta-lactamase PCA consists of fusing complementary fragments of beta-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active beta-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by beta-lactamase.     

Detecting protein-protein interactions using Renilla luciferase fusion proteins

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.     

A LuxP-based fluorescent sensor for bacterial autoinducer II.

Autoinducer 2 (AI-2), which enables different bacterial species to engage in interspecies communication, has been difficult to detect quantitatively. Currently, the most commonly used method for AI-2 detection employs an engineered Vibrio harveyi reporter strain, which produces bioluminescence in response to AI-2. However, the bioassay is not quantitative and is sensitive to assay conditions. In this work, we have developed two protein sensors for AI-2 by modifying AI-2 receptor proteins LuxP and LsrB with environmentally sensitive fluorescent dyes. The protein sensors bind specifically to AI-2 and produce dose-dependent changes in their fluorescence yield. The new assay method has been applied to monitor the enzymatic synthesis of AI-2 in real time and determine the extracellular and intracellular AI-2 concentrations in several bacterial culture fluids.     

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.     

Engineering a high-affinity methyl-CpG-binding protein

Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.     

Bioorthogonal proteomics of 15-hexadecynyloxyacetic acid chemical reporter reveals preferential targeting of fatty acid modified proteins and biosynthetic enzymes

Chemical reporters are powerful tools for the detection and discovery of protein modifications following cellular labeling. The metabolism of alkyne- or azide-functionalized chemical reporters in cells can influence the efficiency and specificity of protein targeting. To evaluate the effect of degradation of chemical reporters of protein fatty acylation, we synthesized 15-hexadecynyloxyacetic acid (HDYOA), a reporter that was designed to be resistant to β-oxidation, and compared its ability to label palmitoylated proteins with an established reporter, 17-octadecynoic acid (ODYA). HDYOA was able to label known candidate S-palmitoylated proteins similarly to ODYA. Accordingly, bioorthogonal proteomic analysis demonstrated that 70% of proteins labeled with ODYA were also labeled with HDYOA. However, the proteins observed differentially in our proteomic studies suggested that a portion of ODYA protein labeling is a result of β-oxidation. In contrast, downstream enzymes involved in β-oxidation of fatty acids were not targeted by HDYOA. Since HDYOA can label S-palmitoylated proteins and is not utilized by downstream β-oxidation pathways, this fatty acid chemical reporter may be particularly useful for bioorthogonal proteomic studies in cell types metabolically skewed toward fatty acid breakdown.     

Ultrasensitive fluorescence-based detection of nascent proteins in gels

The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as [(35)S]methionine or [(14)C]leucine. We report a sensitive, nonisotopic, fluorescence-based method for the detection of nascent proteins directly in polyacrylamide gels. A fluorescent reporter group is incorporated at the N-terminus of nascent proteins using an Escherichia coli initiator tRNA(fmet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, we find that the protein translational machinery accepts BODIPY-FL, a relatively small fluorophore with a high fluorescent quantum yield, as an N-terminal modification. Under the optimal conditions, fluorescent bands from nanogram levels of in vitro-produced proteins could be detected directly in gels using a conventional UV-transilluminator. Higher sensitivity ( approximately 100-fold) could be obtained using a laser-based fluorescent gel scanner. The major advantages of this approach include elimination of radioactivity and the rapid detection of the protein bands immediately after electrophoresis without any downstream processing. The ability to rapidly synthesize nascent proteins containing an N-terminal tag facilitates many biotechnological applications including functional analysis of gene products, drug discovery, and mutation screening.     

Bioluminescent monitoring enables observation of intracellular events in real time without cell and tissue destruction

Secreted reporter proteins provide monitoring of intracellular events in real time without cell destruction. To create human melanoma cell lines that enables noninvasive bioluminescent monitoring of metabolic activity, a comparison of the efficiency of isoforms and mutant variants of luciferase from the Metridia longa as secreted reporter proteins in the cells of human melanoma lines Mel IL was conducted. The MLM3 deletion mutant had the highest activity in the medium of two studied isoforms and two deletion mutants of secreted M. longa luciferase during the Mel IL melanoma cell transfection. It was established that optimization of the gene structure of the selected MLM3 variant for expression in human cells increases the level of bioluminescent activity in the Mel IL cells by almost an order of magnitude. A stable Mel IL melanoma cell line with constitutive expression of the humanized hMLM3 reporter gene was obtained and characterized. The linear range of identification of living cells by the hMLM3 reporter activity was more than three orders of magnitude with a sensitivity of detection of 10 cells.

     

A Fluorescence‐Based Sensor Assay that Monitors General Protein Aggregation in Human Cells

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ‐peptide. Exposure to proteome destabilizers lead to re‐localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide‐genetic screens to identify genes and pathways involved in protein misfolding and aggregation.     

A bichromatic fluorescent reporter for cell-based screens of alternative splicing

Alternative splicing is the primary source of proteome complexity in metazoans and its regulation shapes the proteome in response to shifting physiological requirements. We developed a bichromatic splicing reporter that uses a peculiar feature of some fluorescent protein coding regions to express two different fluorescent proteins from a single alternative splicing event. The mutually exclusive expression of different fluorescent proteins from a single reporter provides a uniquely sensitive approach for high-throughput screening and analysis of cell-specific splicing events in mixed cell cultures and tissues of transgenic animals. This reporter is applicable to the majority of alternative splicing patterns and can be used to quantify alternative splicing within single cells and to select cells that express specific splicing patterns. The ability to perform quantitative single-cell analysis of alternative splicing and high-throughput screens will enhance progress toward understanding splicing regulatory networks and identifying compounds that reverse pathogenic splicing defects.     

Thermostable lichenase as a translational reporter

Hybrid genes containing the reporter gene for thermostable lichenase and model genes recA, recA1, cry3a, cry3aM, and ssp1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research.     

A three-hybrid screen identifies mRNAs controlled by a regulatory protein

RNA-protein interactions are important in many biological contexts. Identification of the networks that connect regulatory proteins to one another and to the mRNAs they control is a critical need. Here, we use a yeast three-hybrid screening approach to identify RNAs that bind a known RNA regulatory protein, the Saccharomyces cerevisiae PUF protein, Mpt5p. The assay selects RNAs that bind in vivo using simple phenotypes and reporter genes. It enables rapid analyses of the affinity and specificity of the interaction. We show that the method identifies mRNAs that are genuinely regulated by the protein in vivo, and that it complements biochemical strategies, yielding a set of mRNAs that overlap with, but are distinct from, those obtained by biochemical means. The approach we describe facilitates construction of protein-RNA linkage maps.     

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.     

Utilization of non-AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

Here we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest. While translation of the reporter is significantly reduced, the levels are sufficient for detection using flow cytometric methods and, in turn, predictive of protein expression from the gene of interest since both ORFs are translated from the same mRNA. Using this system, CHO cells have been sorted to obtain enriched pools producing significantly higher levels of recombinant proteins than the starting cell population and clones with significantly better productivity than those generated from limiting dilution cloning. This method also serves as an effective screening tool during clone expansion to enable resources to be focused solely on clones with both high and stable expression.     

Monitoring protein modification with allosteric ribozymes

An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.     

The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins

The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.