1.3 Purification of Tata-Box Binding Protein (version: 01/21/00)

Protein purification is the essence of biochemistry. Much of the biochemical literature contains descriptions of how specific proteins are separated from the thousands of other proteins and biomolecules in tissues, cells, and biological fluids. Biochemical investigations of biological processes require the isolation, purification, and characterization of proteins. The properties of proteins vary widely and no single technique or sequence of techniques can purify all proteins.

Protein purification can be often simplified by altering the target protein at the DNA level. One can build affinity for a specific small molecule into a protein. The affinity technique used here relies on a Histidine-Tag. Histidine-Tag (His-Tag) purification is a convenient, rapid one-step protein purification method that uses metal chelation. For purification of a His-Tagged protein, metal ions are immobilized by chelation with reactive groups covalently attached to a solid support (resin). A His-Tag (six to ten adjacent histidine residues) has strong affinity for metal ions such as Ni2+. To use this technique one must first isolate the cDNA (coding DNA) of the target protein, and add to that DNA a series of base triplets that each encode for histidine. The altered DNA is inserted into a plasmid overexpression system and use it to transform a strain of E. coli. The E. coli expresses the target protein fused to the histidine tag.

A His-tagged protein can generally be purified in a single chromatographic step because a his-tagged protein, but not other proteins, will bind tightly to a Ni2+-containing resin (nickel agarose). A his-tagged target protein will be selectively retained on a column of nickel agarose, and can be eluted with imidazole. His-tagged proteins, but not other proteins, bind to the immobilized metal on the resin. After washing away other proteins, the his-tagged protein is eluted with imidazole. Imidazole competes with histidine for metal binding. Imidazole concentrations of the wash and elution buffers under native conditions can be adjusted to minimize co-purification of non-specifically bound proteins.

His-Tag Affinity Purification of TBP. The target protein in this experiment is the DNA binding domain of Tata Box Binding Protein (hTBPc),which has been fused at the N-terminus to a His-Tag. Residues 155-335 of human TBP were fused with an additional 20 N-terminal amino acids (MGSSHHHHHHSSGLVPR*GSH N->C). The protease trypsin cuts at *, leaving GSH as the new N-terminus end of the protein. This fusion protein was constructed by R.G. Roeder (Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021).

hTBPc Purification Protocol

Required Solutions

Buffer #1	40mM Tris-HCl pH 8.0	20% glycerol	200mM KCl	2mM MgCl2	5mM imidazole
Buffer #2	40mM Tris-HCl pH 8.0	20% glycerol	1M KCl	2mM MgCl2	5mM imidazole
Buffer #3	40mM Tris-HCl pH 8.0	20% glycerol	200mM KCl	2mM MgCl2	20mM imidazole
Buffer #4	40mM Tris-HCl pH 8.0	10% glycerol	200mM KCl	2mM MgCl2	60mM imidazole
Buffer #5	40mM Tris-HCl pH 8.0	10% glycerol	200mM KCl	2mM MgCl2	250mM imidazole

Lysis of Bacteria

1. The TA will grow a 250 ml prep of E. coli that overproduces His-Tagged hTBPc, and will spin the culture down in a centrifuge, and give you the pellet. Obtain the pellet from the TA. Gently resuspend the pellet in 20ml of Buffer #1. Keep your protein at 4 deg C (on ice) for every step of the purification. If your protein warms up it will be degraded by endogenous proteases.
2. Sonicate the suspension for 4 sets of 10 pulses, keeping the sample on ice the entire time and leaving 1 minute between sets. After sonication the sample should be viscous and uniform and should not contain any clumps.
3. Centrifuge the sample for 20 minutes at 13,000g and 4oC.

Precipitation of DNA with Protamine Sulfate

4. After centrifugation, carefully collect the supernatant. Discard the pellet.
5. With the supernatant on ice, add (dropwise) 2.5ml of 10% protamine sulfate. Protamine is a small highly cationic protein that will precipitate nucleic acids. Continue stirring for 10 minutes after addition of the protamine.
6. Centrifuge for 15 minutes at 13,000g and 4oC.
7. Discard the pellet and place the supernatant into a 50mL falcon tube. Keep on ice. The supernatant contains the protein which you will purify in the next step. Set aside 40 microliters of the supernatant in an eppendorf tube for PAGE Gel analysis. Label this sample as "crude extract".

Nickel Column Purification

8. The TA will provide you with a nickel-agarose column, which has already been charged with NiSO4. Equilibrate the column with Buffer #1 for 10 minutes (see your TA for the appropriate flow rate on your particular pump).
9. After this equilibration step, begin to load your protein sample onto the column. Collect the flow-through and label it FT1. After the sample has been loaded, continue to wash the column with Buffer #1 for 15 minutes.
10. Wash the column for 20 minute with Buffers #2, then #3 and then #4. Collect the flow-throughs and label them FT2, FT3 and FT4, respectively.
11. Begin to wash the column with Buffer #5, which will elute the target protein from the column. Collect the flow-through as 1.5mL samples in eppendorf tubes. Wash the column with Buffer #5 until you have collected 20 samples.

PAGE Gel to Determine Purity of hTBPc

12. Prepare a PAGE Gel according to the protocol given under Analytical Techniques on the course syllabus page.
13. Prepare 9 eppendorf tubes with 35 microliters of each of the following samples: Crude extract, FT1, FT2, FT3, FT4 and fractions 3, 4, 5 and 6 (from Buffer #5 wash).
14. To each tube add 60 microliters of Laemmli sample loading buffer.
15. Load each sample onto the gel. Run the gel for 45 minutes at 45 mAmp (gels from two groups will be run on the same gel).

Silver Stain Visualization

16. Follow the silver staining protocol given under Analytical Techniques on the course syllabus page.
17. Analyze the hTBPc bands to see which fractions should be pooled (check to see if
any of the fractions contain contaminating bands). Pool the fractions containing pure hTBPc.

Bradford Protein Assay

18. Follow the Bradford Assay protocol given under Analytical Techniques on the course syllabus page to determine the concentration of protein in your sample.