Traditional construction of a GAL4-AD fusion protein library for yeast two-hybrid (Y2H) assays entails a lengthy workflow (cDNA synthesis, cloning the cDNA library into a yeast expression vector, library-scale amplification in E. coli, and library-scale transformation into yeast), requiring significant time and effort before screening can even begin. Our Matchmaker systems, including our latest Matchmaker Gold Yeast Two-Hybrid System, are highly optimized tools for screening cDNA libraries in yeast and provide a simple workflow to identify novel DNA-binding proteins or protein-protein interactions (Figure 1).
Figure 1. The Mate & Plate protocol. To screen a Mate & Plate library, an aliquot of the library in the MATαY187 strain is mixed with a bait-expressing, MATa reporter strain culture (Y2HGold or AH109). The two strains are cocultured overnight and then plated on selective agar medium.
Simple screening with Mate & Plate libraries
We offer an extensive variety of validated, pretransformed cDNA libraries in yeast. Our Mate & Plate libraries are cDNA libraries of GAL4-AD prey fusion proteins that are ready for immediate screening in Y2H systems. These libraries have been transformed into the MATα haploid yeast strain, Y187, which can be easily mated to a haploid MATa reporter strain (such as Y2HGold or AH109). We also offer the option to generate your own custom libraries with our Make Your Own "Mate & Plate" Library System.
Our Mate & Plate protocol makes library screening an easy task: simply combine the Mate & Plate library culture with a culture of your bait-expressing reporter strain (Figure 1). Coculturing the two strains overnight produces an array of diploid yeast clones, each coexpressing your bait with a different library prey protein. The clone pool can then be plated on selective media to screen for individual clones that express the appropriate reporter genes and markers, indicating the presence of interacting hybrid protein pairs.
We also offer a selection of normalized Mate & Plate libraries which further simplify the search for novel PPIs. Duplex-specific nuclease (DSN) normalization selectively removes abundant, and therefore redundant, cDNAs from the total pool and enriches the library for less abundant sequences (Figure 2). This process eliminates a major source of potential false positives. Library complexity effectively increases, reducing the number of independent clones that must be screened in order to detect genuine positive interactions. This lowers the frequency of false positives that emerge from primary, low-stringency screens. By using normalized Mate & Plate libraries together with the stringent screening methodology provided by the Matchmaker Gold Yeast Two-Hybrid System, your primary screens will greatly favor the identification of genuine positives, produce few false positives, and yield virtually no background colonies.
Figure 2. Normalization reduces the abundance of cDNAs derived from highly expressed genes. Universal cDNA, synthesized using RNA from mixed human tissues, was analyzed before and after normalization on a NimbleGen Homo sapiens microarray. The data shown represents the 7,000+ genes that exhibited greater than 10,000 Mean Fluorescence Units (MFU). The signal intensities for approximately 3,300 of the most highly represented genes were significantly reduced following normalization, reflecting a preferential reduction of these abundant cDNAs.
To illustrate how DSN normalization results in more balanced gene representation, cDNA samples before and after normalization were compared on a NimbleGen microarray containing 47,633 human genes (Figure 2). In this analysis, cDNA species from the most highly expressed genes were preferentially eliminated, while less abundant cDNAs remained largely unaffected. Consequently, the representation of low-copy-number sequences increased within the total cDNA pool. Genes whose cDNA levels were sharply reduced following normalization included two highly expressed housekeeping genes, ß-actin and GAPDH (Figure 3).
Figure 3. DSN normalization removes highly abundant cDNAs. Normalized (Lanes N) and non-normalized (Lanes C) human HeLa S3 cDNAs were compared using virtual Northern blot analysis and 32P-labeled probes. The levels of these two highly abundant cDNAs were sharply reduced following normalization.
Universal coverage, efficient Y2H screens
Our universal libraries provide the broadest and most complete coverage of genes expressed in almost any tissue. To create these all-purpose, normalized libraries, we combined RNAs from a diverse collection of either mouse or human whole tissues that are specifically chosen to represent an expansive range of expressed genes. These same RNA pools are used for our qPCR Human Reference cDNAand qPCR Human Reference Total RNA, as well as our Mouse Universal Reference Total RNA. Following cDNA synthesis and amplification using SMART technology, we normalize each cDNA pool before constructing and transforming the library into yeast. Combining "across-the-board" gene representation with the enrichment of low-copy-number cDNA, our universal normalized libraries offer the greatest capacity for identifying genuine binding partners of your protein of interest.
To demonstrate this, we searched for binding partners of a murine p53-bait using our Mate & Plate Library - HeLa S3 (Normalized). A medium-stringency screen of only 279,000 clones yielded 62 colonies that could possibly contain p53-binding proteins (Table I). In contrast, we recommend screening 1–2 million clones for a standard library. Of eight colonies that were selected for further analysis, four contained three different, well-known binding partners of p53: PCNA, PRMT3, and PTEN. Thus, it is possible to screen a significantly smaller number of clones in a normalized library and still generate valuable data.
Normalized library
Number of clones screened
279,000
Total number of colonies on medium-stringency selection plates
62
Number of colonies analyzed
8
Verified p53 binding partners
Human proliferating cell nuclear antigen (PCNA; isolated twice)
Human protein arginine methyltransferase 3 (PRMT3)
Human phosphatase and tensin homolog (mutated in multiple advanced cancers 1; PTEN)
Human succinate dehydrogenase complex, subunit B, iron sulfur (lp) (SDHB)
Unknown gene
Table I. Identification of p53 binding partners using our Mate & Plate Library - HeLa S3 (Normalized). Binding partners of a murine p53 bait were initiated using the normalized human HeLa S3 library. Binding partners for p53 were selected for by plating on medium-stringency (SD/–leu/–trp/–his) plates. Colonies were analyzed by performing yeast colony PCR on prey inserts from eight colonies and sequencing PCR products.
Make your own Mate & Plate library
For researchers wishing to construct and transform their own cDNA library, we offer the convenient and straightforward Make Your Own "Mate & Plate" Library System. This system combines SMART technology with highly efficient homologous recombination in yeast, allowing you to efficiently synthesize cDNA and then clone it into thepGADT7-Rec AD Cloning Vectordirectly in yeast. Your library is constructed and transformed into the Y187 Yeast Strain in a single step. The result is a custom-made library ready to be used in our easy Mate & Plate protocol.
Mate & Plate libraries, coupled with the stringent screening of the Matchmaker Gold Yeast Two-Hybrid System, provide the most complete and advanced system for discovering new protein relationships. These tools afford you the greatest opportunities for Y2H screening success with the least amount of time and effort. Finally, to further optimize your workflow, we offer Aureobasidin A—a highly effective, selectable marker—to simply and rapidly screen colonies in Y2H screens.
