TITLE: Parallel rapid expression and purification of proteins for crystallography (PREPX): 48x 100 mL cultures
AUTHORS: michael.fairhead

[DESCRIPTION]
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.



[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 24 well agar plate and incubate 240 min 37 °C*.
 

SECTION: Expression
2. Grow  1.5 mL 240 min in suitable container (2 mL 96-well plate, 15 ml faclon) of each clone in superbroth + 1 % glucose + the appropriate antibiotics.

SECTION: Expression
3. Use 1 mL to inoculate 100 mL 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 in 50 ml falcon tubes and discard supernatant.
SECTION: Expression
7. Harvest remainder of culture in same tube at 4000 x g, 20 min, 4 °C, discard supernatant and freeze pellet in falcon tube at -80 °C.
 


SECTION: Cell lysis
8. Thaw pellets.
9. Add 20 mL Base Buffer to each tube containing 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, 20 millimolar (mM) imidazole.


10. Vortex to dissolve pellet and leave 30 min Room temperature.

11. Add 4 mL 10 % Triton X-100*** to each tube and bring volume to  40 mL using Base Buffer 


 


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
15. Apply supernatant from a single tube to 1 mL His GraviTrap column (Cytiva) fitted with LabMate extender.
 

SECTION: Purification
16. 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