Purification of TFIIIC from yeast

Summary

WCE Extracts are prepared from strain SHY282 (TFC4-Flag-EE (HpaI)).TFIIIC complex is first purified by flag affinity: TFC4 contains a single copy of the flag epitope.The flag purified TFIIIC is then further purified by anion exchange on a Source Q column.

Preparing Yeast Whole Cell Extracts

Yeast Strain: The strain used for this method is SHY282.In this strain,the chromosomal TFC4 had been disrupted with the HIS3 gene.It was then transformed with a centromeric plasmid (pRS314 vector)containing the TFC4 gene tagged with two copies of EE and one flag epitopes inserted at the HpaI site near the N-terminus of TFC4.

For Extract preparation,follow the Hahn Lab Method "Yeast WCE using microfluidizer" with the following modifications and additions:

1.To make saturated culture combine 5 ml of YPD with 25µl of 0.4% adenine and 1 blob of yeast.Incubate at 30℃ for 18 to 24 hr until yeast is at a density of 2.0 x10⁸ cells/ml.

2.Grow cells to an OD₆₀₀ of ~1.0 after inoculating 1 liter of YPD (0.002% Adenine)with 1 ml of saturated culture.Culture in the evening harvest in the morning.

3.2 passes through the microfluidizer.

4.Expect ~2 grams of cells per liter of culture.

5.Resuspend ammonium sulfate pellets in HEG (20 mM HEPES,pH 7.6,1 mM EDTA,10 % Glycerol)buffer rather than Buffer C.

6.Generally,not all of the ammonium sulfate precipitate will redissolve in the HEG buffer.To get rid of this excess precipitate,centrifuge WCE at 14K rpm for 10 minutes after resuspending ammonium sulfate pellets.This centrifugation step can be done before or after dialysis,but before dialysis is recommended since the precipitate may complicate the dialysis.

7.Dialyze samples into HEG buffer + 300 mM KCl.

Flag antibody affinity purification of TFIIIC

Followed Hahn Lab Method "EE antibody affinity purification (yeast TFIID)" with the following modifications and additions:

1.Use HEG buffer throughout the procedure.

1.Use the following binding reaction: 1 part beads,16 parts binding buffer,8 parts whole cell extract.The ratio of beads to WCE is based on a protein concentration of 10 to 15 mg/ml,if the protein concentration is lower or higher adjust the ratio of beads in a similar fashion.

2.For any step using Brij58 or NP40,substitute Tween-20.For the purposes of purification,any of these detergents is acceptable,Brij58 and NP40 are avoided because they may interfere in Pol III immobilized template transcription experiments.

3.Use 0.3 M KCl for binding and washing steps.Use 0.5 M KCl for peptide elution and pre-elution bead equilibration.

4.Bind sample for 2 hours

5.Elute four times with 1mg/ml of flag.Expect ~50% of the TFC4 in the WCE to bind to the beads.Almost all of the TFC4 bound to the beads will elute in the first 3 elutions.

Anion Exchange Purification of TFIIIC

TFIIIC is bound to Source Q at 0.2M KCl and is eluted with a gradient from 0.2 M to 0.5M KCl over 25 column volumes.Use as small a column as possible (0.5 ml or less)to decrease the likelihood of loss by non-specific absorption.TFC4 elutes in two separate peaks.The first peak contains ~30% of the TFC4 and all of the TFIIIC DNA binding activity.The second peak contains ~70% of the TFC4 (which is likely in a complex with a subset of the TFIIIC polypeptides)and no TFIIIC DNA binding activity.

Running Buffer:

20mM Tris (pH8.0)

1mM EDTA

10% Glycerol

0.05% Tween-20

1 mM DTT (= 0.154 mg/ml)

0.5 mM PMSF (= 0.08 mg/ml or 1/200 dilution of 16 mg/ml PMSF in Ethanol)

2mM benzamidine (= 0.31 mg/ml or 1/100 dilution of 31 mg/ml benzamidine)

0.6uM leupeptin (= 0.3μg/ml or 1/500 dilution of 0.15 mg/ml leupeptin in ethanol)

2uM pepstatin A (= 1.4μg/ml or 1/200 dilution of 0.28 mg/ml pepstatin A in methanol)

3.3uM chymostatin (= 2μg/ml or 1/2,500 dilution of 5mg/ml chymostatin in DMSO)

References: Marck,C.,Lefebvre,O.,Carles,C.,Riva,M.,Chaussivert,N.,Ruet,A.& Sentenac,A.(1993).The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs.PNAS 90,4027-31.