Transcription factors and other DNA binding proteins must be characterized to understand genetic regulation, DNA repair, and the life cycle of the cell -- all important goals for the improvement of human health and well-being. Affinity chromatography provides the only rational way of purifying many of these components. Late in the last project period we made three important new discoveries which will be characterized over the next budget period. Here, we improve this chromatography by: 1. Develop CNBr-activated Silica into a usable RPLC support. We recently adapted the cyanogen bromide activation chemistry to silica. This new approach should result in efficient coupling with few chromatographic artifacts 2. Catalytic chromatography will be tested by the resolution of DNA polymerases to answer important questions about the advantages of this chromatographic mode. We recently showed that affinity chromatography using both binding- and catalyticspecificity, i.e., """"""""catalytic chromatography,"""""""" gives superior yield and purity of a restriction enzyme (16). We will learn if catalytic chromatography can perform separations impossible by other means as we suspect, relying on subtle differences in catalytic mechanism. This approach may also selectively purify active enzyme away from inactive forms. 3. A model of catalytic chromatography will be constructed relating retention time, binding affinity, catalytic rate, and protein load. 4. Characterize those conditions that give highest purity and yield with a new """"""""trapping"""""""" protocol affinity chromatography. We have found ways in which very low (nM) concentrations of DNA can be used to """"""""trap"""""""" a particular transcription factor from the complex mixture of DNA-binding proteins in cells. This method has already been shown to give higher purity than other procedures. This new method will be improve by defining those conditions that work best. 5. CNBr-silica and the catalytic and trapping chromatography modes will be combined to test the resolution and other properties of these hybrid technologies. These five years of study will make affinity chromatography less empirical and more reliable, improving progress across a broad cross-section of the biomedical sciences.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043609-13
Application #
6519375
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Edmonds, Charles G
Project Start
1989-08-01
Project End
2005-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
13
Fiscal Year
2002
Total Cost
$214,500
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Jia, Yinshan; Nagore, Linda; Jarrett, Harry (2015) Southwestern Blotting Assay. Methods Mol Biol 1334:85-99
Jia, Yinshan; Jarrett, Harry W (2015) Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose. Anal Biochem 482:1-6
Nagore, Linda I; Jarrett, Harry W (2015) T(3): targeted proteomics of DNA-binding proteins. Anal Biochem 474:8-15
Jia, Yinshan; Larionov, Oleg; Jarrett, Harry W (2014) Coupling of deoxyribonucleic acid to solid supports using 3' terminal ribose incorporation. J Chromatogr A 1339:73-9
Nagore, L I; Nadeau, R J; Guo, Q et al. (2013) Purification and characterization of transcription factors. Mass Spectrom Rev 32:386-98
Hoffmann, Christoph; Zimmermann, Anika; Hinney, Anke et al. (2013) A novel SP1/SP3 dependent intronic enhancer governing transcription of the UCP3 gene in brown adipocytes. PLoS One 8:e83426
Jarrett, Harry W (2012) Proteomic methodologies to study transcription factor function. Methods Mol Biol 786:315-34
Zhou, Yanwen; Jia, Yinshan; Jarrett, Harry W (2012) Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)ýýý-tailed duplex DNA promoter for promoter trapping. Anal Biochem 427:133-8
Jiang, Daifeng; Mummidi, Srinivas; Ahuja, Sunil K et al. (2011) CCR5 promoter haplotype transcription complex characterization. J Health Care Poor Underserved 22:73-90
Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W (2011) Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis. J Chromatogr A 1218:7003-15

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