Core C - Protein Expression and Purification Core The success of the entire Program of Projects hinges on the availability of large amounts of protein reagents as well as on the ability to rapidly clone, express and purify a very diverse set of mutant proteins. For example, hundreds of milligrams of core histones and histone chaperones will be required for the proposed research. Additionally, the Projects demand the flexibility to prepare smaller amounts of specific mutants and truncations of histones and chaperones. Specifically, these reagents will be required for the studies on histone acetylation patterns, histone chaperone interactions, nucleosome dynamics and regulation of higher order chromatin structures. Smaller quantities of highly pure histone chaperones, histone acetyl transferases, transcription factors and coactivators will also be needed. Rigorous quality control of the shared reagents will ensure maximum reproducibility for the research in the three Projects. The Protein Expression and Purification (PEP) Core is fully equipped for the high-throughput preparation of large amounts of proteins. The Director, Dr. Karolin Luger, and the Associate Director, Teri McLain, have many years of experience in the large-scale and small-scale purification of a large variety of proteins. The PEP CORE has the following specific aims:
Aim 1 : Express and purify histones from E. coli.
Aim 2 : Express and purify histone chaperones from E. coli and baculovims-infected Sf9 cells.
Aim 3 : express and purify histone acetyltransferases.
Aim 4 : Express and Purify Activators and Accessory Proteins.
Aim 5 : Extract and Purify Endogenous Proteins from Tissue and Cells.
Aim 6 : Maintain an Up-To-Date Inventory of All Protein Produced by the PEP CORE in the Chromatin Intranet Website.
|Chassé, Maggie H; Muthurajan, Uma M; Clark, Nicholas J et al. (2017) Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin. Methods Mol Biol 1608:231-253|
|White, Alison E; Hieb, Aaron R; Luger, Karolin (2016) A quantitative investigation of linker histone interactions with nucleosomes and chromatin. Sci Rep 6:19122|
|Chen, Xu; D'Arcy, Sheena; Radebaugh, Catherine A et al. (2016) Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the Inducible GAL Locus. Mol Cell Biol 36:1287-96|
|Brehove, Matthew; Wang, Tao; North, Justin et al. (2015) Histone core phosphorylation regulates DNA accessibility. J Biol Chem 290:22612-21|
|Kuo, Yin-Ming; Henry, Ryan A; Huang, Liangqun et al. (2015) Utilizing targeted mass spectrometry to demonstrate Asf1-dependent increases in residue specificity for Rtt109-Vps75 mediated histone acetylation. PLoS One 10:e0118516|
|Mattiroli, Francesca; D'Arcy, Sheena; Luger, Karolin (2015) The right place at the right time: chaperoning core histone variants. EMBO Rep 16:1454-66|
|Chatterjee, Nilanjana; North, Justin A; Dechassa, Mekonnen Lemma et al. (2015) Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF. Mol Cell Biol 35:4083-92|
|Muthurajan, Uma M; Hepler, Maggie R D; Hieb, Aaron R et al. (2014) Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone. Proc Natl Acad Sci U S A 111:12752-7|
|Groocock, Lynda M; Nie, Minghua; Prudden, John et al. (2014) RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response. EMBO Rep 15:601-8|
|Blakeslee, Weston W; Wysoczynski, Christina L; Fritz, Kristofer S et al. (2014) Class I HDAC inhibition stimulates cardiac protein SUMOylation through a post-translational mechanism. Cell Signal 26:2912-20|
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