Purification of Kinesin-1 Motor Domain Protein
| Induced cells (2 – 5 g pellet of pET/KHC in BL21(DE3)pLysS host cells)|
| HEM buffer = ||10 mM HEPES pH 7.2|
1 mM DTT
1 mM MgCL2
1 mM EGTA
| Protease Inhibitor Cocktail (200X) = ||1 microgram/mL Pepstatin A|
1 microgram/mL Leupeptin
2 microgram/mL Aprotinin
2 microgram/mL TAME
| SP Sepharose column (2 cm diameter column containing 2 – 3 mL resin)|
| Q Sepharose column (2 cm diameter column containing 2 – 3 mL resin)|
| Centricon 30 spin concentrator (Amicon) |
| Superose 12 FPLC column (Pharmacia or comparable)|
| Beckman TLX ultracentrifuge and TLA rotor, or comparable |
| Pharmacia or comparable FPLC system |
| 1. ||Induce cells and harvest as described in|
Bacterial Expression of Motors. Wash induced cells with HEM + 25 mM NaCl + 0.5 mM PMSF and freeze in 30 mL Oak Ridge centrifuge tubes at -80°C until use.
| 2. ||Resuspend frozen cells on ice at 1 – 1.25 mL/g of HEM + 25 mM NaCl. Add PMSF to 1 mM and 1/200 x volume protease inhibitors. Resuspend the cells using a glass rod and avoid foaming. |
| 3. ||Freeze/thaw to ensure the cells are lysed by freezing tube in liquid N2 for 3 – 4 min, then swirling in a 37°C waterbath until just thawed and still cold. Transfer to ice. |
| 4. ||Add DTT to 0.5 mM, another aliquot of PMSF and protease inhibitors, MgCl2 to 5 mM and DNAse I to 40 micrograms/mL. Incubate 15 – 20 min on ice to digest DNA. Add another aliquot of PMSF and protease inhibitors halfway through the incubation. |
| 5. ||Centrifuge at 18,000 rpm (39,000 x g) and 4°C for 20 min in the Sorvall SS-34 rotor. |
| 6. ||Transfer clear yellow supernatant to Beckman TLA ultracentrifuge tubes. Centrifuge at 80,000 rpm (270,000 x g) and 2°C for 30 min in the TLA 100.3 rotor in a Beckman TLX ultracentrifuge. |
| 7. ||Apply clear yellow supernatant to SP-Sepharose column at 4°C equilibrated with HEM + 25 mM NaCl. Wash column extensively (~8 mL) with HEM + 25 mM NaCl. |
| 8. ||Elute column with HEM + 100 mM NaCl (5 x 1 mL fractions), then with HEM + 200 mM NaCl (8 x 1 mL fractions). The bulk of the protein starts to elute with 100 mM NaCl in the 2nd fraction. Add 5 microliters of 200 mM PMSF to each peak fraction. |
| 9. ||Pool peak fractions, dilute 1:1 with HEM and load onto a Q Sepharose column equilibrated with HEM + 50 mM NaCl. Wash column extensively with HEM + 50 mM NaCl.|
| 10. ||Elute column with HEM + 100 mM NaCl (3 x 1 mL fractions), then with HEM + 200 mM NaCl (8 x 1 mL fractions). The protein peak elutes with 200 mM NaCl in the 2nd to 5th fractions.|
| 11. ||If further purification is necessary, reduce volume using a Centricon 30 spin column, then add 1 volume HEM to reduce NaCl concentration to 100 mM.|
| 12. ||Apply to Superose 12 FPLC column equilibrated in HEM + 100 mM NaCl. Collect 0.5 mL fractions. Pool peak fractions (fr 26-28). Freeze in liquid N2 and store at -80°C . |
| 1. ||KHC and other kinesin motor proteins bind to SP-Sepharose, while most bacterial proteins flow through. Chromatography on SP-Sepharose is therefore an important purification step and can yield fractions of 90-95% homogeneity. |
| 2. ||The SP Sepharose and Q Sepharose columns can be prewashed with 5 M NaCl and equilibrated in HEM + 25 mM NaCl or HEM + 50 mM NaCl. After each use, wash with 10 – 20 mL HEM + 5 M NaCl followed by 10 – 20 mL of HEM + 25 mM NaCl or HEM + 50 mM NaCl. |
| 3. ||The SP Sepharose and Q Sepharose column fractions can be rapidly assayed by using the Bio-Rad protein concentration reagent to identify the peak fractions. Add 5 microliters of each fraction to 395 microliters HEM + 100 microliters of 1:4 diluted Bio-Rad reagent and read OD595 relative to a control with no added protein. |
| 4. ||The peak fraction from SP Sepharose is ~3 – 5 mg/mL KHC motor domain protein starting from 2-5 g cells. |
H Song & SA Endow 2/99
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Created 2 March 1999 21:00 GMT
Modified 29 October 2004 16:45 GMT