Ni-NTA purification and on-column renaturation of denatured His-tagged nanoCLAMP proteins from NEc1 cell pellets
nanoCLAMPs with an N-terminal His-tag and a C-terminal cysteine are purified from BL21-derivative NEc1 under denaturing conditions with Ni-NTA resin. The denaturant is removed while the protein is bound the column, and proteins are eluted in non-denaturing buffer.
Prepare "Denaturing IMAC Buffer - TCEP"
Make 10 ml per g of cell pellet plus safety
Add 10 ml "Denaturing IMAC Buffer - TCEP" per g of cell pellet and resuspend
The cell pellet is prepared as described in the protocol "Induction of Protein Expression from E. coli NEc-1 - Frozen Stock to Cell Pellet"
Sonicate with a homogenizing tip until the cell pellet is fully resuspended
Typically 2 to 3 minutes. Minimize foaming. Representative set up: Kinematica Polytron PT-10-35 with tip PTA 10 TS, speed <3
Transfer the suspension to a 30 ml Oak Ridge Centrifuge Tubes
Transfer up to 18 ml of suspension per tube to avoid leaking
Centrifuge the suspension in a fixed angle rotor at 30,000 X g for 30 minutes at 15°C
Thermo FiberLite F21-8x50y at 15,900 rpm
During the centrifugation step, transfer the appropriate volume of "Qiagen Ni-NTA resin, 50% slurry" to a 50 ml conical tube
The appropriate volume of resin typically ranges from 0.6 to 1.4 ml per g of cell pellet. The appropriate amount can be determined by small scale titration.
Centrifuge the Ni-NTA resin slurry in a swinging bucket rotor at 1000 X g for 4 minutes
Beckman S4180 at 2,448 rpm
Discard the supernatant, resuspend the resin in 3 bed volumes of "Denaturing IMAC Buffer," and centrifuge in a swinging bucket rotor at 1000 X g. Repeat this step for a total of 3 times
Resuspend the Ni-NTA resin in "Denaturing IMAC Buffer" to make a 50% slurry and then transfer the slurry to a conical tube or chromatography column. Drain or centrifuge to remove excess buffer
Use a container with sufficient volume for the resin and the cleared lysate
Transfer supernatant from the centrifugation of the cell suspension (cleared lysate) to the container containing the Ni-NTA resin
Seal the container of cleared lysate and Ni-NTA resin and rotate for at least for 2 hours at Room Temperature
Binding time can be increased to up to 24 hours
Remove the cleared lysate either by draining the column or centrifuging in a swinging bucket rotor at 1000 X g for 4 minutes
If desired, save the supernatant for later analysis
If the Ni-NTA resin is in a centrifuge tube, add "Denaturing IMAC Buffer - TCEP" to form a slurry. Transfer the slurry to a chromatography column. Wash the centrifuge tube with additional buffer and transfer the wash to the column to maximize recovery
Connect the column to a liquid chromatography system with a flow adapter filled with "Denaturing IMAC Buffer"
Wash the column with 6 bed volumes of "Denaturing IMAC Buffer - TCEP"
For a 1 cm diameter, set the flow rate at 1 ml/min. For a 2.5 cm diameter column, set the flow rate at 2 ml/min.
Wash the column with 6 bed volumes of "Denaturing IMAC Buffer" at the same flow rate
Wash the column with 10 bed volumes of "Non-Denaturing IMAC Buffer" at the same flow rate
Set the chromatography system so that "Denaturing IMAC Buffer" is replaced by "Non-Denaturing IMAC Buffer" in all lines and valves downstream of the column outlet
Reduce the flow rate to half and wash the column with 10 bed volumes of "Non-Denaturing IMAC Buffer" at Room Temperature
For a 1 cm diameter column, set the flow rate at 0.5 ml/min. For a 2.5 cm diameter column, set the flow rate at 1 ml/min.
During the wash, weigh a tube for collecting the eluate
Set the chromatography system to begin peak collection when the A280 exceeds 0.1 and end peak collection when the A280 decreases to 0.16.
Maintain the same flow rate and elute bound protein with "Non-Denaturing IMAC Buffer - 250 mM Imidazole"
Store eluted protein at 4°C
|Use / Nickname||Solution||Usage Rate|
|For lysing the cell pellets||Denaturing IMAC Buffer - TCEP||10 ml per g of cell pellet|
|For equilibrating the Ni-NTA resin||Denaturing IMAC Buffer||10 ml per ml of Ni-NTA resin|
|For transferring the Ni-NTA resin with bound protein and washing the column||Denaturing IMAC Buffer - TCEP||10 ml per ml of Ni-NTA resin|
|For renaturing the denatured protein bound to the Ni-NTA resin||Non-Denaturing IMAC Buffer||20 ml per ml of Ni-NTA resin|
|For eluting the renatured protein bound to the Ni-NTA resin||Non-Denaturing IMAC Buffer - 250 mM Imidazole||10 ml per ml of Ni-NTA resin|
Outside Material(s) Used