Purification of Kar3 Motor Domain Protein

互联网2013-09-06

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Induced cells (2 - 5 g pellet of pET/Kar3 in BL31(DE3)pLysS host cells) (See note #1)

HEM buffer =

10 mM HEPES pH 7.2
1 mM MgCL₂
1 mM DTT
1 mM EGTA

HEM + 80 mM NaCl

HEM + 100 mM NaCl

HEM + 200 mM NaCl

200 mM PMSF in EtOH

Protease Inhibitor Cocktail (200X) =

1 microgram/mL Pepstatin
1 microgram/mL Leupeptin
2 micrograms/mL Aprotinin
2 micrograms/mL TAME

1 M DTT

1 M MgCl₂

10 micrograms/mL DNAse I

SP Sepharose column (2-3 mL bed volume)

Centricon 30 spin concentrator (Amicon)

Mono S column (Pharmacia or comparable)

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 (6 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 can be ~90-95% pure. Add 5 microliters of 200 mM PMSF to each peak fraction.
9.	Pool peak fractions, then add 2 volumes HEM to reduce NaCl concentration to < 70 mM.
10.	Apply to Mono S column equilibrated in HEM. Elute column with a linear gradient of 0 - 500 mM NaCl. The Kar3 protein elutes as a major peak at 200 mM NaCl and a minor peak at 230 mM NaCl. Both peaks consist of highly purified Kar3 motor domain protein by SDS-PAGE.
11.	Minor contaminants present in the major peak of Kar3 from the Mono S column can be removed by chromatography on a Superose 12 FPLC column equilibrated in HEM + 100 mM NaCl. Reduce volume using a Centricon 30 spin column and adjust salt concentration to 100 mM NaCl before loading onto the column. Collect 0.5 mL fractions. Pool peak fractions (fr 26 - 28). Freeze in liquid N₂ and store at -80°C.

1.	The Kar3 motor domain comprises 347 amino acids and corresponds to residues 383-729 of S. cerevisiae Kar3. The coding region of the Kar3 motor domain contains 10 Arg_AGA codons. For expression of protein, Kar3 was cloned into pMW172 (Way et al. 1990) and co-transformed with pACYC184/argU ⁺ (Spanjaard et al. 1990) into competent host cells. argU ⁺ is the E. coli gene for tRNA Arg_AGA , which is limiting in E. coli . The cells are grown in M9ZB medium containing 5 micrograms/mL of tetracycline in addition to ampicilin and CAP.
2.	Kar3 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.
3.	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.
4.	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.

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