Purification of Human K560-GFP molecular motor

pET17b-Kif5b(1-560)-GFP-His should be transformed into BL21(DE3)RIPL cells.

Make enough LB for at least 7.5L of culture.

Grow an overnight starter culture.

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 200rpm until OD600 reaches 0.6-0.8.

Chill cells and incubator to 18°C

Add 0.5millimolar (mM) and allow it to grow at 200rpm

Harvest and freeze cell pellets.

Resuspend 7.5L worth of pellets in 120mL supplemented with 1 cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 50mL . Also add 1mg/mL .

Incubate On ice for 0h 30m 0s .

Lyse the resuspension by sonication.

Add 0.5millimolar (mM) to the sonicate, and clarify by centrifugation 40000rcf,4°C in Type 70 Ti rotor (Beckman).

Incubate the supernatant with Ni-NTA agarose beads incubated with the Wash buffer . Nutate for 1h 0m 0s

Apply to gravity column and wash with 100mL.

Resuspend the beads in elution buffer, incubate for 0h 5m 0s and elute in 0.5mL increments.

Combine peak fractions and buffer exchange on PD-10 desalting column equilibrated with Storage buffer .

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).

A total of 1mL of motor solution from previous steps is used. Incubate with 1millimolar (mM) and 20micromolar (µM) On ice in the dark for 0h 5m 0s and subsequently warm to Room temperature .

Polymerize bovine brain tubulin (about 0h 30m 0s at 37°C) and centrifuge (TLA120.2 rotor at 80000rpm at Room temperature) through a glycerol cushion . Resuspend the pellet.

Incubate the microtubule solution with the resuspended microtubules in the dark for 0h 15m 0s atRoom temperature.

Put the incubated solution on top of glycerol cushion and centrifuge in a TLA120.2 rotor at 80000rpm

at Room temperature.

Wash the final pellet with BRB80 and incubate in 100µL for 0h 5m 0s atRoom temperature .

Centrifuge the kinesin solution 72000rpm in TLA100 rotor at Room temperature .

Collect the supernatant and supplement with 660millimolar (mM) and flash freeze.