Purification of Ncd Motor Domain Protein
| Induced cells (2 – 5 g pellet of pET/Ncd 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)|
| 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 + 80 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 + 80 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 + 80 mM NaCl. Wash column extensively (~8 mL) with HEM + 80 mM NaCl. |
| 8. ||Elute column with HEM + 100 mM NaCl (3 x 1 mL fractions), then with HEM + 200 mM NaCl (7 x 1 mL fractions). The bulk of the protein elutes with 200 mM NaCl in the 2nd to 4th fractions (fr 200-2 to 200-4) and is ~90-95% pure. Add 5 microliters of 200 mM PMSF to each peak fraction. |
| 9. ||Pool peak fractions, reduce volume using a Centricon 30 spin column, then add 1 volume HEM to reduce NaCl concentration to 100 mM. |
| 10. ||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. ||Ncd 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 typically yields fractions of 90-95% homogeneity for the Ncd motor domain protein (residues 333-700). |
| 2. ||The SP Sepharose column can be prewashed with 5 M NaCl and equilibrated in HEM + 80 mM NaCl. After each use, wash with 10 – 20 mL HEM + 5 M NaCl followed by 10 – 20 mL of HEM + 80 mM NaCl. |
| 3. ||The SP 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 ~5 – 10 mg/mL Ncd motor domain protein starting from 2 – 5 g cells. If the protein concentration of the SP Sepharose unbound fraction is < 20 mg/mL, the cells were not lysed properly. |
| 5. ||The Ncd motor domain protein tends to aggregate and should be kept in 50 – 100 mM NaCl during the purification. Partially purified protein undergoes degradation when kept on ice >1 – 2 hrs – the purification should be carried out rapidly and any SP Sepharose column fractions that are saved should be flash frozen in liquid N2 and stored at -80°C . |
| 6. ||Missense mutants containing only a single amino acid change can be dramatically altered in their ability to bind to SP Sepharose. It is therefore necessary to carry out trial experiments for variant or mutant Ncd proteins to determine if the protein binds to SP Sepharose and, if so, the NaCl concentration at which the protein elutes. |
H Song & SA Endow 2/99
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Created 2 March 1999 21:00 GMT
Modified 29 October 2004 16:45 GMT