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Kloning och uttryck av det cellcykel-relaterade proteinet Mus81
Cloning and expression of the DNA repair and cell-cycle associated protein Mus81
Mapping protein-protein interaction networks has come in vogue recently thanks to advancement in experimental techniques, and thanks to the increasing wealth of genomic sequence and protein structure data made available. Protein-protein interactions are important because it is the basis for many of the processes in living cells (for example detection of external signals from the environment, DNA repair, synchronization of the cell-cycle, and many more). High through-put protein interaction techniques such as two-hybrid screening or mass spectrometry (see for example Ho et al) can facilitate identification of many protein interaction pairs, but it does not provide any information on the nature of the interaction; on how the proteins interact on a molecular level. On the other hand, nuclear magnetic resonance (NMR) provides a unique capability to identify the interaction interface between molecules on the level of atomic resolution.
Specific plan and suitable previous knowledge:
Mus81 is a protein recently identified as an important component and interaction hub in the DNA metabolism network (Haber and Heyer). The structure of the protein is known (Nishino et al), but its format of interaction to its various partners remains to be characterized thoroughly. The long-term goal of the project is to clone and express Mus81 and several of its interaction partners and characterize their interactions with NMR and bioinformatics techniques. In the first round a student would clone the mus81 gene, as well as separate domains of the protein, and express the corresponding proteins in E. coli. NMR techniques and biochemical assays would be used to validate protein quality and to characterize the products. Subsequent and/or parallel projects are readily available. Suitable previous experiences include gene cloning, protein purification, biochemical assays, and familiarity with NMR spectroscopy.
Haber and Heyer, Cell 107 (2001), 551-554
Ho et al., Nature 415 (2002), 180-183
Nishino et al, Structure 11 (2003), 445-457
Protein interaction, NMR, gene cloning, protein purification, DNA metabolism, biochemistry.
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