Finding a Mate

Available Two-hybrid Systems Graphic: Leza BerardoneGenome sequencing has produced a vast supply of proteins in need of a functional identity. One way to identify a protein's function is to identify its interacting partners, because proteins often work in pairs or as part of large complexes. Scientists traditionally have used biophysical or biochemical methods (such as affinity chromatography or co-immunoprecipitation) to study protein-protein interactions. More recently, two-hybrid and phage-display systems have dominated the field with clear advantages, the most attractive of which is the immediate accessibility to the protein's genetic information. The two-hybrid system is an in vivo assay, so proteins are more likely to be in their native conformation, and weak and/or transient interactions that may evade detection in vitro can be detected. Conventional two-hybrid systems are based on the fact that many eukaryotic transcriptional activators contain two physically and functionally separate domains, both of which are required for activity. The DNA-binding domain (DBD) binds to specific cis-regulatory sequences, whereas the activation domain (AD) recruits and instructs RNA polymerase II to transcribe downstream genes. While the DBD and AD are generally part of the same protein, as in the native yeast GAL4 protein, they may also function separately provided they are linked in some way that tethers the AD to the promoter. The standard yeast two-hybrid system identifies an interaction between two proteins by reconstituting active GAL4 protein.1 The two proteins are expressed as fusions with the GAL4 DBD and AD domains, so that their interaction links DBD and AD together at the promoter, reconstituting transcriptional activity. This causes reporters that contain GAL4-binding sequences to be transcribed. While the "bait" is usually a specific protein, the "prey/target" can be either another known entity or a cDNA or genomic library. As GAL4 is endogenous to yeast, it may interact with a variety of yeast cell elements; these nonspecific interactions may inadvertently affect yeast cell growth. Thus, false positive results can arise from autoactivation of reporter transcription by the bait protein alone if it contains intrinsic DNA-binding/activating activities. Systems employing the prokaryotic LexA heterologous transcriptional activator are therefore a popular alternative, often yielding fewer false positives. A number of other limitations are inherent to yeast two-hybrid systems. For example, non-nuclear proteins can be difficult to detect; hybrid proteins may not be expressed stably in yeast or may not efficiently move into the nucleus; the creation of hybrid proteins may impede the natural folding patterns of the proteins, or obscure their interaction sites; and yeast cells lack complex post-translational modification mechanisms needed by many proteins for proper function. Improved two-hybrid systems overcome many of these limitations. New and Improved Several new features have been incorporated into yeast two-hybrid systems, such as smaller vectors for increased transformation efficiency, expanded multiple cloning sites, increased reporter numbers for higher-stringency detection and decreased false positive results, reporters with variable sensitivities for fine-tuning detection, protein fusions including epitope tags or nuclear localization signals (NLS), and galactose-inducible expression or low-copy number vectors for use with potentially toxic proteins. The GAL4 Two-Hybrid System 3 from BD Biosciences-CLONTECH of Palo Alto, Calif., contains a number of these improvements. This kit employs high-level protein expression bait and prey vectors that express the proteins of interest as DBD and AD fusions with c-Myc and hemmaglutinin (HA) epitope tags, respectively. Epitope tags provide a convenient way to isolate interacting proteins without the need for specific antibodies to each new protein. Both vectors also contain T7 promoters for direct in vitro transcription and sequencing reactions. System 3 features a new yeast strain, AH109, which contains three reporter genes, each under the control of distinct GAL4 upstream activating sequences (UAS) and TATA boxes. Thus, false positives resulting from nonspecific interactions with flanking sequences or transcription factors are decreased. Yeast strains carrying the bait vector pGBKT7 (used in this system) transform more efficiently than do strains harboring other bait vectors,2 making introduction of AD libraries easier and providing more thorough representation of all library sequences. CLONTECH's new MATCHMAKER Library Construction and Screening kit, which incorporates each of these features, allows cloning by homologous recombination, enabling users to construct and screen a two-hybrid library in a week starting with less than 1 µg of total RNA. The GAL4-based ProQuest Yeast Two-Hybrid System from Rockville, Md.-based Life Technologies, a division of Invitrogen of Carlsbad, Calif., employs low-copy number vectors to decrease nonspecific protein interactions and toxic effects resulting from hybrid protein overexpression. Three reporters with independent and unrelated promoters increase the system's detection stringency and result in four potential phenotypes. ProQuest is ideal for studies aimed at characterizing mutations, peptides, or other compounds that disrupt known interactions for which expression levels closer to physiological conditions are valuable. According to Phil Vanek, Gene Function business unit manager at Life Technologies, the ProQuest System now supports Gateway-compatible two-hybrid libraries, allowing identified targets to be moved quickly and easily into any of several Gateway-compatible destination expression vectors for further analysis. The HybriZAP® 2.1 Yeast Two-Hybrid System from Stratagene of La Jolla, Calif., is another improved GAL4-based system, which uses a lambda vector to generate high-efficiency cDNA and genomic libraries containing a high percentage of inserts and increased representation. The lambda libraries convert easily to plasmid format for two-hybrid screening by in vivo mass excision and replication in a lambda-resistant Escherichia coli host. Stratagene offers a custom library production service for this system. A number of LexA systems are also available. Rockville, Md.-based OriGene's DupLEX-A yeast two-hybrid system employs the Escherichia coli LexA DBD and the "acid blob" protein B42 AD. Three different bait vectors allow the researcher to choose the orientation of the LexA sequences--5' or 3' to the protein of interest--and the option of using a vector containing an NLS. The prey vector is GAL1-inducible and carries an HA tag. The system provides a choice of three different reporters with variable sensitivities containing one, two, or eight LexA sites, thus allowing a mildly autoactivating bait to be used when coupled with a less sensitive reporter. Like most companies, OriGene also offers premade fusion libraries constructed in their prey vector. The LexA-based Hybrid Hunter system from Invitrogen features smaller vectors with expanded multiple cloning sites. The bait vector is selectable in both yeast and bacteria via the novel antibiotic zeocin, providing maximum compatibility with other systems' prey vectors. The prey vector also encodes NLS and V5 epitope tags. Two yeast strains are provided, L40 and EGY48, with the latter offering galactose-inducible expression. Premade libraries with either V5 or HA epitope tags are available separately. Not Just for Yeast Anymore Courtesy of PromegaPromega's Mammalian Two-hybrid system can circumvent some of the problems associated with detecting interactions in yeast. Detection of mammalian protein interactions in yeast may be misleading, as the proteins are not in their native environment and may behave differently. As a result, a number of companies now offer mammalian two-hybrid systems. The CheckMate™ Mammalian Two-Hybrid System from Promega of Madison, Wis. uses a bait vector that expresses Renilla reniformis luciferase, which allows normalization for transfection efficiency, and a firefly luciferase reporter. Reporter and control activities are assayed simultaneously using Promega's Dual-Luciferase® Reporter Assay System, making the system rapid, easy, and sensitive. Control vectors have been proven in five different mammalian cell lines: NIH3T3, CHO, 293, BHK-2, and HeLa. The system's prey vector has an NLS as well as the neomycin resistance gene, which allows for the selection of stable transformants. Stratagene's Mammalian Two-Hybrid Assay Kit, another luciferase-based detection system, has been proven in HeLa, CHO, COS, NIH3T3, and 293 cells. Finally, CLONTECH's Mammalian MATCHMAKER uses a chloramphenicol acetyl transferase (CAT)-based reporter system. Working with E. coli is more time- and cost-efficient than working with yeast, and most scientists are familiar with the molecular biology techniques involved with bacterial use. Also, bacteria can usually be transformed at higher efficiency, permitting the screening of larger, more complex libraries. Stratagene's recently introduced BacterioMatch™ Two-Hybrid System is a rapid, easy alternative or complement to yeast two-hybrid systems and is based on transcriptional activation. The bait is expressed as a fusion with the full-length bacteriophage *cI protein containing the N-terminal DBD and C-terminal dimerization domain. The target protein is fused to the N-terminal domain of the RNA polymerase *-subunit. A bait-prey interaction causes RNA polymerase to bind near the promoter to initiate transcription and express the ampicillin resistance gene, the reporter in this system. ß-galactosidase (LacZ), expressed from the same "activatable" promoter, can be used to identify false positives. Whither Two-Hybrid? Invitrogen's Dual-Bait Hybrid Hunter Yeast Two-Hybrid System is a unique product utilizing two bait constructs and two separate reporter systems for simultaneous detection of two different sets of protein interactions in one screening--a combination that could save considerable time and expense. The system can also be used for competitive screenings involving proteins or compounds that may interrupt interactions. The first bait-prey interaction is selected using leucine prototrophy and a LacZ assay, as in the original Hybrid Hunter, whereas the second interaction is identified via lysine prototrophy and a ß-glucuronidase assay. Carlsbad, Calif.-based Qbiogene's Q-PID Protein Interaction Detector features an RNA polymerase (Pol) III-based detection system as opposed to the more well known Pol II-based systems. Pol III is involved in the transcription of far fewer genes than is Pol II, resulting in decreased background levels. The kit uses a lethal selection method based on toxicity to 5-fluoro-orotic acid (5-FOA). The bait protein is expressed as a fusion with the GAL4p DBD, which binds to a GAL4p upstream activator sequence (UASG). The prey is expressed as a fusion with the *138 transcriptional activator. The reporter, SNR6, is essential for Saccharomyces cervisiae growth. The yeast indicator strain QYN1 contains an SNR6-UASG cassette in place of the chromosomal copy of the SNR6 gene. A second, constitutive, wild-type (WT) copy of SNR6 is present on a plasmid that also carries the conditional lethal gene for orotidine-5'-phosphate-decarboxylase (URA3). When bait and prey interact, the AD recruits Pol III, activating expression from the chromosomal SNR6. Incubation on plates containing 5-FOA screens out cells dependent on the plasmid copy of SNR6 for growth. This system is useful for studying many protein-protein interactions, especially those thought to be involved in the regulation of the Pol II pathway. Most traditional systems are less effective at detecting interactions between cytoplasmic proteins. With the CytoTrap® Two-Hybrid System from Stratagene, the interaction search occurs in the cytoplasm instead of the nucleus. Thus, proteins that depend on post-translational processing in the ER or cytoplasm for function, proteins involved in transcriptional regulation, and those needing cytosolic or membrane entities, are ideal subjects for this system. The CytoTrap system is based on reconstitution of the Ras signaling pathway rather than on transcriptional activation. The system uses the novel yeast strain cdc25H, which contains a mutation in the guanyl nucleotide exchange factor cdc25 gene (the yeast homologue for hSos). Activation of the Ras pathway, and cell growth and survival, are dependent on this protein. The cdc25 mutation in cdc25H is temperature-sensitive, such that cell growth can occur at 25ºC, but not at 37ºC. The hSos gene product can complement the cdc25 gene, permitting growth at 37ºC, as long as hSos is localized to the membrane via a protein-protein interaction. The target protein or cDNA library is expressed as a fusion with the Src myristylation signal, which anchors it to the cytoplasmic surface of the cell membrane. The bait protein is fused with hSos. When bait and target are co-transformed into cdc25H yeast, only colonies containing a specific protein-protein inter-action grow at 37ºC. BioSignal Packard of Montreal, Canada, offers another advancement in protein-protein interaction detection, previously described in The Scientist.3 The Bioluminescence Resonance Energy Transfer (BRET2) system uses Renilla luciferase (Rluc) as the donor and a Green Fluorescent Protein (GFP2) as the acceptor molecule in an assay analogous to Fluorescence Resonance Energy Transfer, but without the need for, or problems associated with, an excitation light source. High-Throughput Screening Assays Two-hybrid systems are now available in high-throughput formats. In fact, genome-wide protein interaction maps are being generated using optimized two-hybrid systems.4 CLONTECH's newly available MATCHMAKER BioSensor Kit can detect two-hybrid interactions in a 96-well plate format within 48 hours using fluorescence technology. Each well of the plate contains a silicone matrix in which a fluorescent dye is embedded. The fluorescence of this dye is quenched by oxygen permeating into the matrix. Yeast cells expressing two interacting proteins are added to a well containing a two-hybrid selection medium. A positive interaction results in yeast cell growth, accompanied by oxygen consumption and dye fluorescence. A correlation can be made between yeast cell growth and fluorescent intensity using any standard fluorescence plate reader. Tiina Sepp, product manager at CLONTECH, emphasizes that BioSensor is intended to be a secondary screening tool whose high-throughput design is optimized for drug development projects. Courtesy of Applied BiosystemsApplied Biosystems' ICAST Technology A completely new screening technology, Intra-cistronic Complementation Analysis Screening Technology (ICAST), is now offered as a service by Applied Biosystems of Foster City, Calif. It is a live-cell functional assay that directly quantitates protein interactions in mammalian cells without regard to cellular localization. The assay utilizes intracistronic complementation of LacZ. Restoration of LacZ activity can occur only when the two fragments of the enzyme are expressed as chimeras with interacting proteins, as the fragments have too low an affinity for each other to form a stable complex otherwise. The assay is highly sensitive and aimed at high-throughput drug discovery programs. Scientists at Applied Biosystems fuse customer-supplied cDNAs with the two pieces of the LacZ gene and create a novel cell line expressing both chimeras that is then delivered to the customer. Even easier is Qbiogene's MERLIN Two-Hybrid Custom Service, which provides a complete outsourced two-hybrid analysis. Researchers can provide Qbiogene with an unamplified two-hybrid library, yeast host strain(s), PCR primers to library vector, and the bait vector containing insert. Qbiogene's scientists perform a yeast two-hybrid screening within a 14-week turnaround time. Researchers then receive plates containing positive yeast colonies, purified plasmid DNA from positive clones, single-pass sequence information from positive clones, and a detailed Certificate of Analysis. After successfully identi-fying and confirming an exciting protein-protein interaction, an additional layer of complexity can be included using CLONTECH's pBridge Three-Hybrid vector to study ternary complexes. This vector, which contains two distinct multiple cloning sites, replaces the DBD vector from the company's System 3 Two-Hybrid kit. The third protein may act to help stabilize two otherwise weakly interacting proteins, it may modify or inhibit other interacting pairs, or as its name indicates, it can act as bridge between two proteins that do not interact directly. Expression of the third protein is regulated by a conditional methionine promoter. Barbara A. Cunningham (Barbara.A.Cunningham@hitchcock.org) is a freelance writer in Lyme, N.H. References 1. S. Fields, O.-K. Song, "A novel genetic system to detect protein-protein interactions," Nature, 340:254-6, 1989. 2. Louret et al., "MATCHMAKER Two-Hybrid System 3," CLONTECHniques, Jan. 1999. 3. J. Piper, "BioSignal Packard's better BRET," The Scientist, 14[24]:25, Dec. 11, 2000. 4. P. Legrain, L. Selig, "Genome-wide protein interaction maps using two-hybrid systems," FEBS Letters, 480:32-6, Aug. 25, 2000.

Finding a Mate

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