TITLE: Parallel rapid expression and purification of proteins for crystallography (PREPX): large scale 1 L cultures
AUTHORS: michael.fairhead

[DESCRIPTION]
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.




[GUIDELINES]
Method overview

Standard workflow is expression via autoinduction followed by purification using IMAC/PD-10/revIMAC and serial gel filtration

[STEPS]
SECTION: Expression
1. Either transform BL21 (DE3) with the appropriate plasmid OR streak from glycerol stock onto agar plate and incubate 240 min 37 °C*.
 

SECTION: Expression
2. Grow 10 mL 240 min in 50 mL tube of each clone in superbroth + 1 % glucose + the appropriate antibiotics.

SECTION: Expression
3. Use 10 mL to inoculate 1 L AIM-TB (+ Antibiotics + Antifoam 204) in a baffled flask.
SECTION: Expression
4. Grow 250 rpm, 240 min, 37 °C shaking using loose foil cover**.
AERATION IS ESSENTIAL!.

 

SECTION: Expression
5. Grow 40-48 h 250 rpm, 18 °C shaking.

SECTION: Expression
6. Harvest at 4000 x g, 20 min, 4 °C.
SECTION: Expression
7. Scrape out pellet and place in plastic polygrip bag and freeze -80 °C.
 


SECTION: Cell lysis
8. Place polygrip bag on flat surface and smash cell pellet into small pieces and pour into 500 mL beaker.
9. Add 3 mL Base Buffer/g cell pellet (10 millimolar (mM) HEPES, 500 millimolar (mM) NaCl, 5 % Glycerol, 0.5 millimolar (mM) TCEP, pH 7.5) + 0.5 mg/mL Lysozyme, 1 μg/ml Benzonase or 10 μg/ml DNase I, 1 % Triton X-100***, 20 millimolar (mM) imidazole.

 

11. Leave 30 min Room temperature.

10. Use stripette to dissolve pellet and put up to 45 mL in a 50 mL tube (4 tubes in total).

12. Freeze -80 °C 1-2 h or overnight if preferred.
13. Thaw in Room temperature water bath 60 min and mix.

14. Centrifuge 4000 x g, 60 min, 4 °C. 
 

SECTION: Purification
16. Apply SN from 2 x 50 mL tubes to 1 mL His GraviTrap column (Cytiva) fitted with LabMate extender.
 

SECTION: Purification
15. Wash 10 mL Base Buffer + 20 millimolar (mM) Imidazole**.
 

SECTION: Purification
17. Slot His GraviTrap column into PD10 column (Cytiva) fitted with LabMate extender (pre-equilibrated in Base Buffer + 20 millimolar (mM) Imidazole).

SECTION: Purification
18. Elute protein with 2.5 mL of Base Buffer + 500 millimolar (mM) Imidazole directly onto PD10 column.

SECTION: Purification
19. Remove His GraviTrap column.
SECTION: Purification
20. Place PD10 into 50 mL falcoln tube and add 3.5 mL Base Buffer + 20 millimolar (mM) Imidazole and collect.

SECTION: Purification
21. Measure A280.
SECTION: Purification
22. Add protease 1 OD unit TEV for every 10 OD units target and incubate 60 min 4 °C.
 



SECTION: Purification
23. Run back over His GraviTrap column equilibrated in Base Buffer + 20 millimolar (mM) Imidazole.

SECTION: Purification
24. Wash column 2.5 mL 20 millimolar (mM) Imidazole.

SECTION: Purification
25. Check purity of 6 mL pool.

SECTION: Purification
26. Concentrate to 1 mL ish.

SECTION: Purification
27. Transfer to 1.6 mL glass autosampler vial ensure at least 1.1 mL in vial!.
SECTION: Purification
28. Run through serial gel filtration system injecting 1 mL.

SECTION: Purification
29. Take peak fraction(s) only (1-2 mL) and concentrated to 10-20 mg/mL if possible.
SECTION: Column regeneration: PD-10
30. Wash PD-10 columns with 50 mL-100 mL of Milli-Q water.

SECTION: Column regeneration: His GraviTrap
32. Wash IMAC columns 40 mL Milli-Q.

SECTION: Column regeneration: His GraviTrap
35. Wash IMAC columns 10 mL 1 Molarity (M) NaOH.

SECTION: Column regeneration: His GraviTrap
34. Wash IMAC columns 40 mL Milli-Q.

SECTION: Column regeneration: His GraviTrap
37. Wash IMAC columns 10 mL 1 Molarity (M) Acetic Acid + 1 % Triton X-100.

SECTION: Column regeneration: His GraviTrap
36. Wash IMAC columns 40 mL Milli-Q.

SECTION: Column regeneration: His GraviTrap
33. Wash IMAC columns 10 mL 20 % Ethanol + 0.1 Molarity (M) EDTA*.

*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!

SECTION: Column regeneration: His GraviTrap
38. Wash IMAC columns 40 mL Milli-Q.

SECTION: Column regeneration: His GraviTrap
39. Wash IMAC columns 0.5 mL 100 millimolar (mM) Nickel Sulfate + 20 millimolar (mM) Tris.HCl pH 8*.
 

SECTION: Column regeneration: His GraviTrap
40. Wash IMAC colums 40 mL Milli-Q.

SECTION: Column regeneration: His GraviTrap
41. Store all columns in water at 4 °C. For long term storage use 20 % Ethanol



SECTION: Column regeneration: PD-10
31. Store all columns in water at 4 °C. For long term storage use 20 % Ethanol