Purification of KHC Motor Domain Protein

互联网2013-09-06

830

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

HEM + 25 mM NaCl

HEM + 50 mM NaCl

HEM + 100 mM NaCl

HEM + 200 mM NaCl

200 mM PMSF in EtOH

Protease Inhibitor Cocktail (200X) =

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

1 M DTT

1 M MgCL₂

10 mg/mL DNAse I

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.