Transcriptional regulation by transcription factors is key to gene expression and the cell s biology. Purification of these proteins is approached empirically. Hundreds of transcription factors are known but few are well characterized. One of the most powerful techniques available for the purification of these proteins is DNA-affinity chromatography. The proposed work will develop a rational approach to transcription factor purifications using high performance DNA-affinity chromatography.
The specific aims of this proposal are: 1.To discover which chromatographic supports provide the best performance in the research laboratory. (Three different supports will be considered: Sepharose, HEMA and silica. The Sepharose will be used as the historical standard since most previous work was done with a Sepharose support. The other two are more modern high efficiency supports.) 2. To determine the preferred method for attaching DNA to a support. (There are numerous chemical means of attaching DNA to the supports. Depending on the site of attachment, the chemical means sometimes inactivates a part of the attached DNA for interaction with the DNA binding proteins. Enzymatic attachment of the DNA to the support is a method developed by the investigators. For some DNA-bind proteins, they have found this to be the preferred method. However, the complexity of the attached DNA is typically by necessity greater when the enzymatic attachment method is used. Therefore nonspecific interactions between the DNA and contaminant proteins from the cells can be a problem with enzymatic attachment.) 3. To determine the DNA sequence which gives optimal performance. (There are various reports that suggest that short pieces of DNA are better for the purification of DNA proteins. However, other reports indicate that long repeats of recognition sequences are optimal.) 4. To optimize the elution protocol for the column. A range of variables have been used to elute DNA-binding proteins from DNA-affinity columns. These include changing the salt concentration, changing the temperature, adding an organic solvent, adding metal ions , and adding a competing DNA. 5. To apply each of these techniques to three different classes of DNA binding proteins. All of the initial optimization work will involve the purification of the lac repressor protein. This protein is the most studied transcription regulator. It has the helix-turn-helix, the C/EBP protein has the leucine zipper motif and the TFIIIA has the Zn-finger motif. After the optimization of the conditions for the lac proteins, the other two proteins will be taken through the same sort of rigorous optimization. 6. Finally, the understanding gained from this will be used to purify the B3 activator protein.
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