Parallel rapid expression and purification of proteins for crystallography (PREPX): 48x 100 mL cultures
michael fairhead
Abstract
This protocol details the parallel rapid expression and purification of 24x proteins for crystallography (PREPX) at 100 mL 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 24 well agar plate and incubate 4h 0m 0s 37°C*.
Grow 1.5mL 4h 0m 0s in suitable container (2 mL 96-well plate, 15 ml faclon) of each clone in superbroth + 1 % glucose + the appropriate antibiotics.
Use 1mL to inoculate 100mL 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 in 50 ml falcon tubes and discard supernatant.
Harvest remainder of culture in same tube at 4000x g,4°C, discard supernatant and freeze pellet in falcon tube at -80°C.
Cell lysis
Thaw pellets.
Add 20mL Base Buffer to each tube containing 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, 20millimolar (mM) imidazole.
Vortex to dissolve pellet and leave 0h 30m 0s Room temperature.
Add 4mL 10 % Triton X-100*** to each tube and bring volume to 40mL using Base Buffer
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
Apply supernatant from a single tube to 1 mL His GraviTrap column (Cytiva) fitted with LabMate extender.
Wash 10mL Base Buffer + 20millimolar (mM) Imidazole**.
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
