Purification of Villin From Chicken Intestinal Epithelium
References:

Bretscher, A. & K. Weber (1980) Cell 20, 839-847

Bretscher, A. (1981) PNAS 78, 6859-6853

I. Needed Items & Solutions:

Dnase affinity column	100% saturated Ammonium Sulfate
column, 1.5 x 20 cm	fraction collector
Centriprep concentrator	Soln.I from brush border prep., 2 L
EDTA/EGTA, 4 liters	0.1 M PMSF
1 mg/ml Aprotinin	1 M DTT

 

Buffer 1:	Buffer 2:	Buffer 3:	Extraction Buffer:
0.15 M KCl	0.6 M KCl	0.15 M KCl	5 mM CaCl2 in
1 mM MgCl2	1 mM MgCl2	1 mM MgCl2	Solution I with
5 mM CaCl2	5 mM CaCl2	5 mM EGTA	0.2 mM PMSF & 1 mg/
20 mM imidazole	20 mM imidazole	20 mM imidazole	liter aprotinin
pH 7.0	pH 7.0	pH 7.0	Room Temperature!
500 ml	200 ml	100 ml	100 ml

All buffers contain 1 mM DTT, 0.1 mM PMSF, & 1 mg/liter aprotinin

II. Extraction of Villin from Brush Borders:

1. Isolate intestinal epithelial cells from 3-4 chickens.

2. NOTE: All procedures are performed at 4 C, or on ice unless otherwise stated. Also, add 0.1 mM PMSF, 0.5 mg/liter aprotinin and 0.2 mM DTT to all solutions.

3. Follow the same protocol as for tropomyosin isolation through homogenization and the first spin. The pellet after the first spin consists predominantly of nuclei and brush borders.

4. Wash soluble cytosolic contaminants from brush border pellet by resuspending pellets in 1 liter of EDTA/EGTA. This should be split into 6, 250 ml centrifuge bottles. Spin down at 3000 rpm in the GSA rotor for 15 min.

5. Repeat the wash of the brush border pellet for a total of 3-4 washes. Once the supernatant is clear, proceed to step 6.

6. Resuspend the brush border pellets in 400 ml Soln. I (75 mM KCl, 1 mM MgCl₂, 1 mM EGTA, & 10 mM Imidazole) and spin @ 3000 rpm for 15 min. in 2 buckets.

7. Villin extraction: discard supernatant and resuspend each pellet in 25 ml of extraction buffer (5 mM CaCl₂ in Soln. I at room temperature). Combine the two solutions, add a stir bar and stir at room temperature for 15 min. The total extraction time should be 20 minutes at room temperature.

8. Remove the stir bar and place in centrifuge tubes. If myosin is to be extracted from these pellets, do not spin above 3000 x g. If villin is the only protein to be isolated, you can go directly to the high speed spin in step 9. Spin the extract at 5000 rpm in an SS-34 rotor or 4,000 rpm in the GSA rotor for 20 min.

9. Carefully remove the supernatant, place in high speed centrifuge tubes and spin at 100,000 x g for 30 min. in the ultracentrifuge. Remove the supernatant with a 9" dispo pipet being careful to avoid the pellet. Measure the supernatant volume in a graduated cylinder. Pour into a 250 ml GSA bucket and add 100% saturated ammonium sulfate (SAS) to achieve a final concentration of 60% SAS. Mix gently and let sit on ice for >45 minutes.

10. Spin down proteins precipitated by 60% SAS at 12,000 rpm in the GSA rotor for 30 minutes. Carefully pour off supernatant.

11. Place the centrifuge bottle in ice at an angle with the pellet side up to drain for 2-3 minutes and remove the collected liquid with a pipet.

12. Rotate the bottle pellet down and with a dispo pipet add 3 pipet fulls (about 5 mls) of extraction buffer to the pellet. Since the pellet will be spread up the side of the bottle, wash the pellet with the buffer from the top down, trying not to pipet up any solid pellet or to make bubbles. Once you can no longer see the slight refraction of the ammonium sulfate pellet up the side, resuspend the pellet by pipetting the solution up and down to break up the bigger chunks. When dissolved, transfer to a 10 ml ultracentrifuge tube; rinse the bottle with fresh buffer and mix it with the rest of the protein.

13. Clarify the sample by spinning at 150,000 x g for 30 minutes. Carefully remove the supernatant. Dilute with extraction buffer to a final volume of 25 ml. Remove a 40 ul aliquot as a gel sample.

III. DNase affinity column purification of villin

1. Decant the DNase affigel into a sidearm vacuum flask and rinse with Buffer 1. Place under a vacuum and continue until no more bubbles appear...about 10 minutes.

2. Chill the DNase affigel on ice: extra buffer should be removed so that only a 1:1 gel:buffer ratio remains. In the cold room pour the gel into a 1.5 x 20 cm column with about 5 mm of buffer in the bottom. Turn on the column and pack under a steady flow of buffer. Once the gel is packed, equilibrate the column by washing with 250 ml of Buffer.

3. Load the crude villin (step 13 above) onto the DNase column at a flow rate of 30 ml/ hour. Collect 5 ml fractions throughout.

4. Wash with 50 ml of Buffer 1.

5. Wash with 250 ml of Buffer 2.

6. Elute villin with 100 ml of Buffer 3. Villin binds to actin in the presence of calcium, but is released in buffer 3 due to the EGTA.

7. Obtain an OD 280 profile and remove 45 uL aliquots from pertinent fractions to mix with 15 ul of 4X sample buffer; run a microgel to see which fractions contain villin.

8. Meanwhile, clean the DNase column.

9. Since villin elutes in only a few fractions, be judicious about combining fractions and save time. Usually only 4-5 fractions contain villin. Combine these fractions, and rinse out the test tubes with a little (0.5 ml) Buffer 3.

10. Concentrate the villin using a Centriprep (Amicon) concentrator to a final volume of about .75 ml. Rinse the concentrator after removal of villin. Store purified villin on ice.

11. Determine the villin concentration using the Lowry assay or OD 280 after dialysis. Villin E = 123.5 mM-1cm-1.

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