TITLE: Purification of Human K560-GFP molecular motor
AUTHORS: Andrea M. Dickey

[DESCRIPTION]
K560-GFP purification protocol from the Reck-Peterson Lab based on protocol from Nicholas et al. 2014. Edited for protocols.io by Andrea Dickey and Mariusz Matyszewski.

[GUIDELINES]
All protein purification steps should be performed at 4 °C unless noted otherwise.


[STEPS]
SECTION: Expression
1. pET17b-Kif5b(1-560)-GFP-His should be transformed into BL21(DE3)RIPL cells.
SECTION: Expression
2. Make enough LB for at least 7.5 L of culture.

SECTION: Expression
3. Grow an overnight starter culture.
SECTION: Expression
4. Transfer starter culture into LB. Make sure to add proper antibiotics (Ampicillin for the plasmid, and Chloramphenicol for the cells we used).
Allow it to grow at   until OD600 reaches 0.6-0.8.

SECTION: Expression
5. Chill cells and incubator to 18 °C 

SECTION: Expression
6. Add 0.5 millimolar (mM) and allow it to grow at   

SECTION: Expression
7. Harvest and freeze cell pellets.
SECTION: Purification
8. Resuspend 7.5 L worth of pellets in 120 mL supplemented with 1 cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 mL . Also add 1 mg/mL .
Incubate on ice for 30 min .

SECTION: Purification
9. Lyse the resuspension by sonication.
SECTION: Purification
10. Add 0.5 millimolar (mM) to the sonicate, and clarify by centrifugation 40000 rcf, 60 min, 4 °C in Type 70 Ti rotor (Beckman).

SECTION: Purification
11. Incubate the supernatant with Ni-NTA agarose beads incubated with the Wash buffer. Nutate for 60 min 

SECTION: Purification
12. Apply to gravity column and wash with 100 mL. 

SECTION: Purification
13. Resuspend the beads in elution buffer, incubate for 5 min and elute in 0.5 mL increments. 

SECTION: Purification
14. Combine peak fractions and buffer exchange on PD-10 desalting column equilibrated with Storage buffer.
SECTION: Purification
15. Peak fractions of the motor solution were either flash-frozen at -80 °C  until further use or immediately subjected to microtubule bind and release purification (see next steps).

SECTION: Microtubule bind and release purification
16. A total of 1 mL of motor solution from previous steps is used. Incubate with 1 millimolar (mM) and 20 micromolar (µM) on ice in the dark for 5 min and subsequently warm to Room temperature .

SECTION: Microtubule bind and release purification
17. Polymerize bovine brain tubulin (about 30 min  at 37 °C) and centrifuge (TLA120.2 rotor at 80000 rpm, 12 min at Room temperature) through a glycerol cushion. Resuspend the pellet.
SECTION: Microtubule bind and release purification
18. Incubate the microtubule solution with the resuspended microtubules in the dark for 15 min atRoom temperature.

SECTION: Microtubule bind and release purification
19. Put the incubated solution on top of glycerol cushion and centrifuge in a TLA120.2 rotor at 80000 rpm, 12 min 
at Room temperature.

SECTION: Microtubule bind and release purification
20. Wash the final pellet with BRB80 and incubate in 100 µL for 5 min atRoom temperature .

SECTION: Microtubule bind and release purification
21. Centrifuge the kinesin solution 72000 rpm, 7 min in TLA100 rotor at Room temperature .

SECTION: Microtubule bind and release purification
22. Collect the supernatant and supplement with 660 millimolar (mM) and flash freeze.