Parallel rapid expression and purification of proteins for crystallography (PREPX): large scale 1 L cultures
michael fairhead
Abstract
This protocol details the parallel rapid expression and purification of proteins for crystallography (PREPX) at a 1 L culture scale. Recombinant proteins are expressed in Escherichia coli using the autoinduction method and then purified in parallel using a IMAC, desalt, tag cleavage, reverse IMAC and gel filtration work flow.
Attachments
Steps
Expression
Either transform BL21 (DE3) with the appropriate plasmid OR streak from glycerol stock onto agar plate and incubate 4h 0m 0s 37°C*.
Grow 10mL 4h 0m 0s in 50 mL tube of each clone in superbroth + 1 % glucose + the appropriate antibiotics.
Use 10mL to inoculate 1L AIM-TB (+ Antibiotics + Antifoam 204) in a baffled flask.
Grow 250rpm,37°C shaking using loose foil cover**.
AERATION IS ESSENTIAL!.
Grow 40-48 h 250rpm,18°C,0h 0m 0s shaking.
Harvest at 4000x g,4°C.
Scrape out pellet and place in plastic polygrip bag and freeze -80°C.
Cell lysis
Place polygrip bag on flat surface and smash cell pellet into small pieces and pour into 500 mL beaker.
Add 3mL Base Buffer/g cell pellet (10millimolar (mM) HEPES, 500millimolar (mM) NaCl, 5 % Glycerol, 0.5millimolar (mM) TCEP, 7.5) + 0.5mg/mL Lysozyme, 1μg/ml Benzonase or 10μg/ml DNase I, 1 % Triton X-100***, 20millimolar (mM) imidazole.
Use stripette to dissolve pellet and put up to 45mL in a 50 mL tube (4 tubes in total).
Leave 0h 30m 0s Room temperature.
Freeze -80°C 1-2 h or overnight if preferred.
Thaw in Room temperature water bath 1h 0m 0s and mix.
Centrifuge 4000x g,4°C.
Purification
Wash 10mL Base Buffer + 20millimolar (mM) Imidazole**.
Apply SN from 2 x 50 mL tubes to 1 mL His GraviTrap column (Cytiva) fitted with LabMate extender.
Slot His GraviTrap column into PD10 column (Cytiva) fitted with LabMate extender (pre-equilibrated in Base Buffer + 20millimolar (mM) Imidazole).
Elute protein with 2.5mL of Base Buffer + 500millimolar (mM) Imidazole directly onto PD10 column.
Remove His GraviTrap column.
Place PD10 into 50 mL falcoln tube and add 3.5mL Base Buffer + 20millimolar (mM) Imidazole and collect.
Measure A280.
Add protease 1 OD unit TEV for every 10 OD units target and incubate 1h 0m 0s 4°C.
Run back over His GraviTrap column equilibrated in Base Buffer + 20millimolar (mM) Imidazole.
Wash column 2.5 mL 20millimolar (mM) Imidazole.
Check purity of 6mL pool.
Concentrate to 1mL ish.
Transfer to 1.6 mL glass autosampler vial ensure at least 1.1 mL in vial!.
Run through serial gel filtration system injecting 1mL.
Take peak fraction(s) only (1-2 mL) and concentrated to 10-20 mg/mL if possible.
Column regeneration: PD-10
Wash PD-10 columns with 50mL-100mL of Milli-Q water.
Store all columns in water at 4°C. For long term storage use 20 % Ethanol
Column regeneration: His GraviTrap
Wash IMAC columns 40mL Milli-Q.
Wash IMAC columns 10mL 20 % Ethanol + 0.1Molarity (M) EDTA*.
*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!
Wash IMAC columns 40mL Milli-Q.
Wash IMAC columns 10mL 1Molarity (M) NaOH.
Wash IMAC columns 40mL Milli-Q.
Wash IMAC columns 10mL 1Molarity (M) Acetic Acid + 1 % Triton X-100.
Wash IMAC columns 40mL Milli-Q.
Wash IMAC columns 0.5mL 100millimolar (mM) Nickel Sulfate + 20millimolar (mM) Tris.HCl pH 8*.
Wash IMAC colums 40mL Milli-Q.
Store all columns in water at 4°C. For long term storage use 20 % Ethanol
