Laboratory of David Burgess
Purification of Intestinal Epithelial Cell Myosin
References:
T.D. Pollard (1982) Methods in Cell Biology 24, 333-371

J.H. Collins & C.W. Borysenko (1984) J. Biol. Chem. 259, 14128-14135


I. Solutions & Equipment:

Columns:

1. 2.5 x 90 cm Sephacryl S-400, equilibrated in column buffer, which takes ca. 2 days of constant flow.
2. Hydroxylapatite column, Biogel HTP, 3 g will result in about a 10 ml column bed.

Solutions:

S-400 buffer: EDTA/EGTA: 4 liters
0.6 M KCl  
1 mM EDTA Myosin Extraction buffer: 100 ml
5% sucrose (w/v) 0.6 M KCl
10 mM imidazole pH 7.5 1 mM MgCl2
1 mM DTT10 mM imidazole pH 7.5 5 mM ATP
0.2 mM PMSF 10% sucrose (w/v)
  0.2 mM PMSF

Solution I:

1 liter 1 mg/liter aprotinin
  1 mM DTT


HTP column buffer: 1 liter  
0.5 M KCl elution buffers:
1 mM EDTAHTP 0.15 M K2HPO4 in HTP buffer:100 ml
1 mM DTT 0.2 M K2HPO4 in HTP buffer: 200ml
10 mM imidazole pH 7.2 0.4 M K2HPO4 in HTP buffer: 100ml
1 mM Na2HPO4 Adjust pH of each to 7.2 with HCl

II. Column Preparation:

1. Sephacryl S-400:

a. This column is stored in the following buffer:

0.6 M KCl

1 mM EDTA

10 mM Imidazole pH 7.5

0.02% NaN3


To fully equilibrate the column in the new buffer, approximately 5 column volumes of freshly made buffer must be run through the column. For a 500 ml column, 2.5 liters of buffer wash is required. A typical flow rate for this column is 45 ml/hour. Thus, 56 hours or 2.5 days prior to use of the column, begin the equilibration wash.

b. Since the column buffer contains 5% sucrose and no antibiotics, I prefer to make fresh buffer each day from dry salts to avoid microbial growth. Also, it is imperative to run 2.5 liters of the above storage buffer into the column immediately after the proteins have eluted from the column. Often I follow the sucrose buffer with 100 ml of 4 M Guanidine:HCl and then the storage buffer to help remove denatured proteins which stick in the column bed.

c. Two hours before the column is to be loaded, begin a zone of 75 ml of Myosin extraction buffer into the column. If you are late in starting the column, just shut off the column and start flow again as you load your sample.

2. Hydroxylapatite, Biogel HTP:

a. Hydration: Biogel HTP is a powder hydroxylapatite which should be hydrated the day the column is to be used. Briefly the hydration procedure is as follows.

  1. Into a 50 ml erlenmeyer flask weigh 3 g of HTP.
  2. Add 20 ml of HTP column buffer without DTT and suspend particles by gently swirling.
  3. Heat 15 min. in a hot water bath, 80-90 C swirling 3 times to suspend particles.
  4. Place in ice and fill to top of flask with HTP column buffer.
  5. Let settle for 10 minutes, pour off or suction off liquid.
  6. Resuspend HTP in column buffer, fill flask to top and let settle again. This is to remove fine particles.
  7. Remove all the liquid above the settled particles. Add and equal volume of HTP buffer to make a 1:1 suspension. Pour column with all components at 4 C. The column itself should have 1 cm of HTP buffer in it.
  8. Let the gel settle in the column for 5 minutes, then turn on the flow to pack the column. When only 3-4 mm of buffer remains above  the top of the column, start washing with HTP column buffer to equilibrate the column. Do not wash with more than 10 column volumes. Volume = r2h, volume in mls, height & radius in cm.
III. Extraction of myosin from brush borders:

NOTE: All procedures are performed on ice; include 0.1 mM PMSF, 1 mg/liter aprotinin, and 1 mM DTT in all buffers. During homogenization and extraction of proteins include twice the concentrations of protease inhibitors. Myosin is susceptible to proteolysis, and the major problem in this protocol is to prevent protease cleavage of the heavy and light chains. Thus, be especially careful to keep all solutions cold and work with the prep. on ice. Although this protocol does not include diisopropylfluorophosphate (DFP), this inhibitor is included at 1 mM in the homogenization and extraction steps in our lab.

1. Isolate intestinal epithelial cells from 2-4 chickens.

2. Homogenize cells in EDTA/EGTA; pellet brush borders and nuclei @ 1500g for 15 min. (3000 rpm in GSA).

3. Resuspend pellets in 6, 250 ml bottles in EDTA/EGTA; spin 1500g for 15 min.

4. Repeat EDTA/EGTA washes until the supernatant is clear.

5. Resuspend pellets in Soln. I & pellet at 1500g for 10 min.

6. Myosin can be extracted after villin extraction but villin must be extracted before myosin extraction. Be sure to perform the low speed spin in the villin prep., since extraction of myosin from the high speed pellet is slower and more difficult due to the hard pellet.

7. Weigh centrifuge bottle containing pellet. Place pellet in a Dounce homogenizer, weigh empty bottle and add ca. 1 ml of Extraction Buffer per gram of brush borders. Do not exceed 20 ml volume, as only 25 ml total volume can be loaded onto a 500 ml column. After 10 passes with the pestle, immediately spin @ 100,000g for 30 min. (try for <10 minutes from homogenization to spinning rotor).

8. Remove supernatant carefully with a pipet and load immediately onto the S-400 column which has a 75 ml zone of Myosin extraction buffer in it. Flow rate should be about 45 ml/hour.

9. Collect 5 ml fractions. The void volume of the column is ca. 25% of the bed volume. Myosin is slightly included, and should elute within ca. 40% of the column volume. If myosin is extracted from whole brush borders, 110K-calmodulin will also be extracted from the microvilli and elutes from the S-400 column in ca. 50% of the column volume. If villin is extracted first from brush borders, 110K-CaM is solubilized with the villin and will not be found in the myosin prep.

10. The myosin peak is usually 45-50 ml in volume; determine the OD 280 profile of the fractions and run microgels of the fractions 40-70. On my 500 ml column myosin elutes between fractions 45-54. Remove 45 ul aliquots from alternate fractions, mix with 15 ul of 4x sample buffer and run as much as can be loaded in each well.

11. Myosin at this stage is usually 95% pure. However, there are some high molecular weight contaminants which can be removed by chromatography on hydroxylapatite. If you choose to further purify myosin, skip the rest of this section and go on to the HTP column.

12. Combine fractions containing myosin, about 50 ml. Dialyze in a 23 mm width dialysis tubing against 2 liters of 5 mM MgCl2, 10 mM Imidazole buffer pH 7.2 & 1 mM DTT. A visible precipitate will form within 6 hours.

13. Collect myosin by spinning at 25,000g for 30 min. (15,000 rpm in SS-34 or 12,000 rpm in an HB-4 swinging bucket rotor).

14. Redissolve myosin pellet on ice in the following buffer:

30% glycerol

0.6 M KCl Determine myosin concentration:
20 mM Imidazole pH 7.6 @ 280 nm, E = 0.56 mg/ml-1 cm-1

1 mM EDTA

1 mM DTT


15. Store myosin by freezing dropwise in liquid nitrogen. Store below -70o C as the melting point is below -20o C.

III. Hydroxylapatite Purification of Myosin:

1. Hydrate, pour & equilibrate HTP column the day it is to be used.

2. Pool the fractions containing myosin from the S-400 column and load them onto the equilibrated hydroxylapatite column. Collect 5 ml fractions throughout.

3. Wash the column with 80 ml of HTP column buffer.

4. Wash the column with 100 ml of 0.15 M KHPO4/buffer.

5. Wash the column with 100 ml of 0.2 M KHPO4/buffer

6. Elute myosin with 100 ml of 0.35 M KHPO4/buffer

7. Determine the OD 280 elution profile

8. Remove aliquots, mix with 4X sample buffer and run a microgel of pertinent fractions.

9. Combine fractions containing myosin, dialyze & store as described in steps 12-15 above.

10. Perform ATPase assays of unfrozen myosin and a frozen sample to compawith the ATPase activity after freezing & thawing the same day the prep is finished.

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