Abstract
The Yeast Two-Hybrid (Y2H) system is the most frequently used method for identifying protein–protein interactions. The use of recombination-amenable Y2H vectors would reduce time and effort for cloning prey or bait vectors, and increase the quality of Y2H screenings due to the production of improved screening libraries. These libraries can heighten the amount of new candidates in Y2H screenings significantly by representing more correct candidate genes in frame and outperform a classical Y2H library. The described vectors can be used for the construction of genomic, peptide, or cDNA-based Y2H libraries. Furthermore, the compatibility to newer ORFeome libraries is also given. Here, we describe a vector system for site-specific recombination and for the construction of high-content Y2H libraries. In summary, we will describe the construction of these vectors and the production of Y2H screening libraries.
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Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–6.
Maier, R., Brandner, C., Hintner, H., Bauer, J., and Onder, K. (2008) Construction of a reading frame-independent yeast two-hybrid vector system for site-specific recombinational cloning and protein interaction screening. Biotechniques 45, 235–44.
Brandner, C. J., Maier, R. H., Henderson, D. S., Hintner, H., Bauer, J. W., and Onder, K. (2008) The ORFeome of Staphylococcus aureus v 1.1. BMC Genomics 9, 321.
Rual, J. F., Hirozane-Kishikawa, T., Hao, T., Bertin, N., Li, S., Dricot, A., Li, N., Rosenberg, J., Lamesch, P., Vidalain, P. O., Clingingsmith, T. R., Hartley, J. L., Esposito, D., Cheo, D., Moore, T., Simmons, B., Sequerra, R., Bosak, S., Doucette-Stamm, L., Le Peuch, C., Vandenhaute, J., Cusick, M. E., Albala, J. S., Hill, D. E., and Vidal, M. (2004) Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res. 14, 2128–35.
Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–95.
Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S., and Vidal, M. (2000) GATEWAY® recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328, 575–92.
Bernard, P., Kezdy, K. E., Van Melderen, L., Steyaert, J., Wyns, L., Pato, M. L., Higgins, P. N., and Couturier, M. (1993) The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. J. Mol. Biol. 234, 534–41.
Lynch, M., Scofield, D. G., and Hong, X. (2005) The evolution of transcription-initiation sites. Mol. Biol. Evol. 22, 1137–46.
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Maier, R.H., Maier, C.J., Önder, K. (2011). Construction of Improved Yeast Two-Hybrid Libraries. In: Lu, C., Browse, J., Wallis, J. (eds) cDNA Libraries. Methods in Molecular Biology, vol 729. Humana Press. https://doi.org/10.1007/978-1-61779-065-2_5
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DOI: https://doi.org/10.1007/978-1-61779-065-2_5
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