About Myriad’s ProNet Program
Myriad’s ProNet program provides researchers the ability to rapidly identify large numbers of novel protein-protein interactions. Since the function of many proteins is mediated by protein-protein contacts, knowledge of these interactions furthers the understanding of the biological role of each interacting partner. Characterization of the networks of protein interactions allows the researcher to:
- Ascribe functions to novel proteins,
- Identify new functions for known proteins,
- Associate proteins with disease pathways,
- Identify new disease genes,
- Determine the interactions that define the critical components in a pathway.
The Technology
The underlying technology for Myriad’s ProNet program is the yeast two-hybrid system. This methodology provides a rapid and accurate way to identify and study protein interactions in vivo. To use this system, two hybrid proteins are generated in a yeast reporter strain (see Figure 1). One of these hybrids consists of the fusion of a site-specific DNA-binding domain to one test protein (the “bait”); the other hybrid consists of the fusion of a transcriptional activation domain to another test protein (the “prey”). These protein constructs are expressed in a yeast reporter strain. If the test proteins interact, they form a functional transcription factor (consisting of both a DNA-binding and an activation domain) that turns on the expression of one or more reporter genes.
To begin a two-hybrid search, a bait construct is prepared by amplification of a gene fragment that encodes a protein or protein domain. This fragment is inserted into a DNA-binding domain vector and used to transform a yeast strain. Libraries of cDNA fragments are prepared in an activation domain vector and are carried in another yeast strain that is of opposite mating type. Yeast carrying a specific DNA-binding domain fusion are mated with yeast carrying an activation domain library and are plated on media that selects for expression of the two-hybrid reporter genes. After incubation, yeast that carry a pair of interacting protein fusions will grow on the selective media and form colonies. These colonies are picked and the insert in the activation domain vector is identified by sequence analysis.
Myriad has taken the yeast two-hybrid methodology and created an industrial-scale system for the rapid discovery of protein-protein interactions. Myriad’s two-hybrid advantages include:
- A proprietary bait creation process that has been fully automated making it reliable and high-throughput,
- Each step in the two-hybrid search process is tracked via an interactive database,
- Custom activation domain libraries have been generated in a proprietary manner to contain few false positives,
- An efficient yeast mating strategy that permits the thorough screening of activation domain libraries.
These skills coupled with Myriad’s proven bioinformatics and sequencing capabilities provide an industrial platform for the discovery and analysis of protein interactions.
Activation Domain Libraries
The activation domain libraries are prepared in vectors designed for the expression of activation domain fusions. The libraries are prepared in the yeast reporter strain that is used for two-hybrid analyses. Every ProNet activation library is created to contain greater than 5 million independent clones so that proteins encoded by low abundance messages are adequately represented. Because the cDNAs are directionally cloned into the activation domain vector, library complexity is increased. Additionally, these libraries are prepared from randomly primed cDNA to eliminate bias towards the 3′ end that is typical with conventional oligo dT priming. Finally, the average insert size is between 0.5 and 1.0 kb because smaller protein fragments typically work better than very large fragments in the two-hybrid system.
ProNet Capabilities
Beginning with proteins of interest a number of bait constructs are generated. These baits are used to screen appropriate activation domain libraries for interacting proteins. The entire process, from bait design to the identification of protein partners, takes about 4-5 months. It is reasonable to expect, on average, several protein partners for each starting protein. As these interacting partners are identified, the scientists involved in the project select some for further analysis by ProNet. By this iterative process a network of protein-protein interactions is developed.
In a typical ProNet project, a set of proteins involved in a particular biological process are selected. These proteins are used to design baits for two-hybrid searches. These baits are screened against appropriate activation domain libraries. Libraries can be custom built from mRNA expressed in specific tissues or cell lines of interest. After protein partners are discovered for the starting set of baits, the partner proteins are used to design a new set of baits to screen. In this manner, a network, or web, of interactions that begins with a desired set of proteins is produced (see Figure 2). The proteins in this network are all likely to be involved in the same biological pathway in the cell.
Bioinformatics
The ProNet database has been specifically designed to facilitate tracking of samples and integration of the discovered protein interactions and novel genes. Information and data from external sources, e.g. public sequences, published yeast two-hybrid protein interactions, and genetic loci, are linked into the data structure. A browser-based interface allows for easy, cross-platform access to the protein network and associated information. A Java-based graphical representation of the derived protein interaction network provides both a mechanism to view the relationships within entire biological pathways, as well as immediate retrieval of specific information about nucleotide sequences, putative identifications, known biological functions, and domain structures, among others.
ProNet Applications
ProNet has the capacity to discover protein-protein interactions regardless of the source of the proteins. For example, a Drosophila gene can be used as the bait and, depending on the cDNA library, human proteins can be found which have the ability to bind to it. Although this would not occur in nature it would be suggestive of a human homolog for the Drosophila protein. The system is equally applicable to human, microbial, veterinary, and agricultural research. Myriad believes that ProNet has applications in the following areas:
- functional analysis of genes identified by various genomics technologies including positional cloning, and differential display;
- extension of biochemical pathways once genes have been identified by EST sequencing, positional cloning, or differential display;
- identification of protein ligands for cell surface receptors;
- identification of receptors for extracellular (secreted) proteins;
- identification of proteins involved in host/pathogen interactions, e.g. viruses, bacteria, and fungi;
- development of biochemical pathways within pathogenic organisms;
- identification of gene candidates for mutation screening;
- identification of peptides that bind to protein targets; and
- identification of targets for small molecule drug development; and
|