Fluorogen-activating scFv biosensors target surface markers on live cells via streptavidin or single-chain avidin.

<p>Fluorescence biosensors are indispensable tools for understanding protein behavior and function in cells. Recent advancements utilize fluorogen-activating-proteins (FAPs) that form complexes with small organic molecules (fluorogens) and result in their fluorescence activation. The technology has found multiple uses in protein discovery applications; however, the current method of detection requires the expression of FAPs as gene fusion tags in cells-a process that is time- and labor-intensive. In this report, we present an alternate method that utilizes FAPs as affinity reagents. Accordingly, we isolated soluble reagents based on FAP fusions with streptavidin (Strep) or avidin proteins, both highly selective for biotin. When tested in vitro, the reagents displayed bi-functional activity, fluorogen activation, and biotin affinity. For live-cell protein discovery, surface targets were biotinylated via biotin-tagged immunoglobulins or a genetically encoded biotin acceptor peptide. As a result, when the cells were labeled with FAP-Strep or FAP-avidin reagent, the in vivo fluorescence measurements indicated high target specificity, minimal background, and bright signal detection. In summary, we present a novel FAP reagent platform that offers a rapid and efficient approach for cell surface protein detection.</p

Fluorogen-activating-proteins as universal affinity biosensors for immunodetection.

<p>Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins-Protein A or Protein G-and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching.</p

Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.

We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein-coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ∼ 1200 off-patent drugs was screened against cells expressing FAP-tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification.</p

High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology.

<p>Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.</p

Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors.

<p>The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g., EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.</p

Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells.

This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2010;00:000-000).</p

A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families.

<p>Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a non-canonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.</p

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

<p>G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.</p

Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate.

The Src tyrosine kinase associates with the focal adhesion adaptor protein Cas (Crk-associated substrate) to suppress the expression of potential tumor suppressor genes. For example, Src utilizes Cas to suppress the expression of the LIM-only protein Fhl1 (four and a half LIM domains 1), in order to promote non-anchored tumor-cell growth and migration. Here, we report that the promoter region of the Fhl1 gene was methylated more in Src-transformed cells than non-transformed cells. In addition, global expression analysis indicates that Fhl1 induced expression of serum deprivation response factor (Sdpr) in Src-transformed cells. Moreover, Fhl1 and Sdpr was expressed in approximately 87% and 40% of samples obtained from non-transformed breast, 100% of samples obtained from non-transformed kidney, and over 60% of samples obtained from non-transformed prostate. In contrast, Fhl1 and Sdpr was detected in approximately 40% and 7% of matched samples from mammary carcinoma, less than 11% of matched samples from kidney carcinoma, and in less than 22% of matched samples from prostate carcinoma. These data indicate that Fhl1 and Sdpr expression was significantly reduced in tumors of the breast (P < 0.02 and P < 0.001), kidney (P < 0.01), and prostate (P < 0.05). In addition, although Src can activate mitogen-activated protein kinase (MAPK) to promote tumor-cell growth, our data indicate that Src did not rely on MAPK activity to suppress the expression of Fhl1 and Sdpr in transformed cells. Thus, Src induced methylation of the promoter region of the Fhl1 gene; Src suppressed Fhl1 and Sdpr expression independent of mitogen-activated protein kinase (MAPK) activity; Fhl1 induced the expression of Sdpr in Src-transformed cells; and Fhl1 and Sdpr expression was suppressed in tumors of the breast, kidney, and prostate.</p

Detection and assignment of mutations and minihaplotypes in human DNA using peptide mass signature genotyping (PMSG): application to the human RDS/peripherin gene.

Peptide mass-signature genotyping (PMSG) is a scanning genotyping method that identifies mutations and polymorphisms by translating the sequence of interest in more than one reading frame and measuring the masses of the resulting peptides by mass spectrometry. PMSG was applied to the RDS/peripherin gene of 16 individuals from a family exhibiting autosomal dominant macular degeneration. The method revealed an A-->T transversion in the 5' splice site of intron 2 that is the likely cause of the disease. It also revealed four different minihaplotypes in exon 3 that represent particular combinations of SNPs at four different locations. This study demonstrates the utility of PMSG for identifying and characterizing point mutations and local minihaplotypes that are not readily analyzed by other approaches.</p