Abstract: Strategy of Purification of the Glutamine-rich Domain of the Corepressor Groucho (2011 SACNAS National Conference)

Saturday, October 29, 2011

Hall 1-2 (San Jose Convention Center)

Kenny Robles , Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA

Alberto Ponce , Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles

Wiam Turki-Judeh, PhD , Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles

Pak Wong , Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles

Albert J. Courey, PhD , Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles

Drosophila Groucho (Gro) is the founding member of a conserved metazoan global corepressor with homologs in vertebrates including humans. Gro family members are characterized by a conserved Glutamine (Q) domain at the N-terminus. Gro facilitates a myriad of biological processes that are essential for normal growth and development and their misregulation can lead to disease. As a corepressor, Gro is recruited to the template by interactions with DNA-bound repressors. Once recruited, Gro represses in a manner that is dependent upon Gro self-association via the Q domain. There are no crystal structures of full-length Gro nor any biophysical data that can link its structure to its ability to repress. This study aims to purify the Gro Q domain to reveal its structure. Our approach is to purify the Q domain by fusing it to Glutathione-S-Transferase (GST), an affinity tag that also increases the solubility of the Q domain. We used anion exchange chromatography to further purify our protein by removing any trace contaminants. The objective is to purify approximately 500uM of the Q domain to use for Hydrogen Nuclear Magnetic Resonance (H-NMR) to reveal its structure. Isotope labeling of the Q-domain using N15 and C13 will complement the H-NMR to solve the structure. These structural findings will give evidence for the role of the Q domain in self-association and, perhaps, an insight into the mechanisms of Gro-mediated repression.