May 23, 2022
Purification of Human K560-GFP molecular motor
- Andrea M. Dickey1
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
Protocol Citation: Andrea M. Dickey 2022. Purification of Human K560-GFP molecular motor. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61xrdvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59048
Keywords: single molecule, kinesin, purification, microtubule, ASAPCRN
Abstract
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.
Materials
Materials:
cOmplete EDTA-free protease inhibitor cocktail tablets
lysozyme
PMSF
Buffers:
Lysis buffer:
- 50 millimolar (mM) Tris pH 7.5
- 300 millimolar (mM) NaCl
- 5 millimolar (mM) MgCl2
- 0.2 Molarity (M) sucrose
- 1 millimolar (mM) DTT
- 0.1 millimolar (mM) Mg-ATP
- 0.5 millimolar (mM) Pefabloc
Wash buffer:
- 50 millimolar (mM) Tris pH 7.5
- 300 millimolar (mM) NaCl
- 5 millimolar (mM) MgCl2
- 0.2 Molarity (M) sucrose
- 10 millimolar (mM) imidazole
- 1 millimolar (mM) DTT
- 0.1 millimolar (mM) Mg-ATP
- 0.5 millimolar (mM) Pefabloc
Elution buffer:
- 50 millimolar (mM) Tris pH 8.0
- 300 millimolar (mM) NaCl
- 5 millimolar (mM) MgCl2
- 0.2 Molarity (M) sucrose
- 250 millimolar (mM) imidazole
- 5 millimolar (mM) bME
- 0.1 millimolar (mM) Mg-ATP
Storage buffer:
- 80 millimolar (mM) PIPES pH 7.0
- 2 millimolar (mM) MgCl2
- 1 millimolar (mM) EGTA
- 0.2 Molarity (M) sucrose
- 1 millimolar (mM) DTT
- 0.1 millimolar (mM) Mg-ATP
Glycerol cushion:
- 80 millimolar (mM) PIPES pH 7.0
- 2 millimolar (mM) MgCl2
- 1 millimolar (mM) EGTA
- 60 % volume glycerol
- 20 micromolar (µM) Taxol
- 1 millimolar (mM) DTT
BRB80:
- 80 millimolar (mM) PIPES pH 7.0
- 2 millimolar (mM) MgCl2
- 1 millimolar (mM) EGTA
- 300 millimolar (mM) KCl
- 7.5 millimolar (mM) Mg-ATP
Expression
Expression
pET17b-Kif5b(1-560)-GFP-His should be transformed into BL21(DE3)RIPL cells.
Make enough LB for at least 7.5 L 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 200 rpm, 37°C until OD600 reaches 0.6-0.8.
Chill cells and incubator to 18 °C
Add 0.5 millimolar (mM) IPTG and allow it to grow at 200 rpm, 18°C, 18:00:00
Harvest and freeze cell pellets.
Purification
Purification
2h 35m
2h 35m
Resuspend 7.5 L worth of pellets in 120 mL Lysis buffer supplemented with 1 cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 mL of Lysis buffer . Also add 1 mg/mL lysozyme .
Incubate On ice for 00:30:00 .
30m
Lyse the resuspension by sonication.
Add 0.5 millimolar (mM) PMSF to the sonicate, and clarify by centrifugation 40000 rcf, 4°C, 01:00:00 in Type 70 Ti rotor (Beckman).
1h
Incubate the supernatant with Ni-NTA agarose beads incubated with the Wash buffer. Nutate for 01:00:00
1h
Apply to gravity column and wash with 100 mL Wash buffer .
Resuspend the beads in elution buffer, incubate for 00:05:00 and elute in 0.5 mL increments.
5m
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).
Microtubule bind and release purification
Microtubule bind and release purification
44m
44m
A total of 1 mL of motor solution from previous steps is used. Incubate with 1 millimolar (mM) AMP-PNP and 20 micromolar (µM) Taxol On ice in the dark for 00:05:00 and subsequently warm to Room temperature .
5m
Polymerize bovine brain tubulin (about 00:30:00 at 37 °C ) and centrifuge (TLA120.2 rotor at 80000 rpm, 00:12:00 at Room temperature ) through a glycerol cushion. Resuspend the pellet.
42m
Incubate the microtubule solution with the resuspended microtubules in the dark for 00:15:00 atRoom temperature .
15m
Put the incubated solution on top of glycerol cushion and centrifuge in a TLA120.2 rotor at 80000 rpm, 00:12:00
at Room temperature .
12m
Wash the final pellet with BRB80 and incubate in 100 µL of release buffer for 00:05:00 atRoom temperature .
5m
Centrifuge the kinesin solution 72000 rpm, 00:07:00 in TLA100 rotor at Room temperature .
7m
Collect the supernatant and supplement with 660 millimolar (mM) sucrose and flash freeze.
Note
A typical kinesin prep in the lab yielded 0.5 micromolar (µM) to 1.5 micromolar (µM) K560-GFP dimer.