A Novel Screen Identifying Ternary Binding Partners Of Know Interacting Proteins
Here, we present the first screening system in yeast that permits high-throughput screening of cDNA libraries for ternary binding partners of a known interaction couple. This is achieved by a unique combination of SUB and yeast mating. Technical prerequisites are special “2in1”-vectors, which allow simultaneous transformation of “Bait I” and “Bait II” on a single plasmid in yeast of one mating type and the cDNA library in the other.
Many biological processes depend on oligomeric protein-protein interactions (PPI). However, state-of-the-art in vivo PPI techniques focus on analysing binary interactions (i.e. the Split-Ubiquitin System (SUS)1). The SUS can also be used to analyse binding of three proteins in the so-called SUS Bridge Assay (SUB)2 . Nevertheless, this assay makes an unbiased screening approach cumbersome and its low efficiency restricts the identification of meaningful candidates.
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The search for potential, yet unknown, third protein binding partners (to two already known proteins) within a given cDNA library by using the SUS Bridge Assay (SUB) requires a rather cumbersome cloning protocol step by step for each given cDNA-clone (transformation of individual plasmids for each assumed cDNA element). In the day-to-day routine of labs this is only feasible for a few preselected proteins, not for a screening of a whole cDNA library.
To enable the screening of a whole cDNA-library we combine the genes for two already known Bait-proteins of a SUS (Bait I and Bait II) in a new 2in1-vector. Transformation of a so called Mat a-yeast strain with this 2in1 vector renders the possibility of using another complementary yeast-strain, a so-called Mat alpha strain, as a vehicle for a whole cDNA library. The scientist can transform any given cDNA library into this second yeast strain. Thus a yeast mating based approach becomes possible. After mating of Mat a and Mat alpha, yeast clones that are expressing protein complexes with at least three interacting and binding partners will render a positive signal.
The mating-based approach is facilitated through simultaneous cloning of both bait proteins on one plasmid backbone and the cDNA library on a second; each of which is either transformed in mating type a or alpha haploid yeast cells, respectively. As proof of concept we have identified novel interaction partners of the brassinosteroid receptor complex of Arabidopsis thaliana.
- Easy identification of still unknown interacting protein binding partners to two already known proteins, this becomes part of the daily routine in laboratories
- Reliable: the new screen identifies real trimeric interaction partners as verified by our data
- Efficient: the mating-based approach guarantees 10-100 times higher coverage of primary interactions compared to standard approaches
- Low-cost: libraries can be re-used and stored in yeast, ready-to-use
Scope of application
- cDNA libraries from virtually all species can be tested
- The system can be expanded to identify quarternary interactions
License or sale for commercial use
collaboration for further development
Publications & Links
1. C. Grefen, P. Obrdlik, K. Harter. The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS). (2009) Methods Mol Biol. 479:217-33
2. A. Honsbein, S. Sokolovski, C. Grefen, P. Campanoni, R. Pratelli, M. Paneque, Z. Chen, I. Johansson, M.R. Blatt. A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis. (2009) In: Plant Cell 21(9):2859-77
Further files for download
- 376 Ki
- DE pending
- PCT pending
Keywordsprotein-protein binding, Split-Ubiquitin-System (SUS), Screening System, cDNA-library, protein interaction, trimeric binding assay, 2in1 vector
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