Parallel rapid expression and purification of proteins for crystallography (PREPX): large scale 1 L cultures

michael fairhead

Published: 2024-01-30 DOI: 10.17504/protocols.io.4r3l22jkxl1y/v1

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

nyikb5id7.docx

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*.

Note

* Freshly transformed or re-streaked cells always give better yields than growing overnights directly from frozen glycerol stocks.

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!.

Note

**An upturned 500 mL plastic beaker with a 2 mL microcentrifuge tube taped to the side of the flask to act as a spacer can also be used.**A breathable membrane such as an AirOtop enhanced flask seal may also be used.

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.

Note

Final wet cell weight is typically 50 g/L of culture

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) + 0.5 Lysozyme, 1 Benzonase or 10 DNase I, 1 % Triton X-100***, 20millimolar (mM) imidazole.

Note

***Triton x-100 is currently restricted for use in the EU and cannot be used without an exemption certificate REACH Annex XIV (Jan 2021). It can be readily subsituted with IGEPAL CA-630 (which is likely to be subject to the same restrictions in the near future). Alternatives that also maybe used are Tergitol 15-S-9 or Tween-20 or octyl glucoside.

10.

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

11.

Leave 0h 30m 0s Room temperature.

12.

Freeze -80°C 1-2 h or overnight if preferred.

13.

Thaw in Room temperature water bath 1h 0m 0s and mix.

14.

Centrifuge 4000x g,4°C.

Note

Higher speed centrifugation can also be performed if desired, e.g. 20,000 g but transfer to suitable centrifuge tubes will be necessary.

Purification

15.

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

Note

Imidazole concentration can be increase to 40 mM in most cases, but may affect yield.

16.

Wash 10mL Base Buffer + 20millimolar (mM) Imidazole**.

Note

**10 mL of a 40 mM or 70 mM imidazole wash can also be done, but this is very target dependent and may lead to significant reduction in final yield BUT can also increase purity substantially, worth trying if your purity is poor.

17.

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

18.

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

19.

Remove His GraviTrap column.

20.

Place PD10 into 50 mL falcoln tube and add 3.5mL Base Buffer + 20millimolar (mM) Imidazole and collect.

21.

Measure A280.

22.

Add protease 1 OD unit TEV for every 10 OD units target and incubate 1h 0m 0s 4°C.

Note

Some targets exhibit significant affinity for IMAC columns even after TEV cleavage try increasing the imidazole concentration to 40 or 70 mM or use an MBP-TEV construct so that the protease can be removed using an amylose column rather than reverse IMAC.

23.

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

24.

Wash column 2.5 mL 20millimolar (mM) Imidazole.

25.

Check purity of 6mL pool.

26.

Concentrate to 1mL ish.

27.

Transfer to 1.6 mL glass autosampler vial ensure at least 1.1 mL in vial!.

28.

Run through serial gel filtration system injecting 1mL.

29.

Take peak fraction(s) only (1-2 mL) and concentrated to 10-20 mg/mL if possible.

Column regeneration: PD-10

30.

Wash PD-10 columns with 50mL-100mL of Milli-Q water.

31.

Store all columns in water at 4°C. For long term storage use 20 % Ethanol

Column regeneration: His GraviTrap

32.

Wash IMAC columns 40mL Milli-Q.

33.

Wash IMAC columns 10mL 20 % Ethanol + 0.1Molarity (M) EDTA*.

*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!

34.

Wash IMAC columns 40mL Milli-Q.

35.

Wash IMAC columns 10mL 1Molarity (M) NaOH.

36.

Wash IMAC columns 40mL Milli-Q.

37.

Wash IMAC columns 10mL 1Molarity (M) Acetic Acid + 1 % Triton X-100.

38.

Wash IMAC columns 40mL Milli-Q.

39.

Wash IMAC columns 0.5mL 100millimolar (mM) Nickel Sulfate + 20millimolar (mM) Tris.HCl pH 8*.

Note

*PUT NICKEL WASTE IN APPROPRIATE CONTAINER FOR DISPOSAL!

40.

Wash IMAC colums 40mL Milli-Q.

41.

Store all columns in water at 4°C. For long term storage use 20 % Ethanol

Inferring cellular and molecular processes in single-cell data with non-negative matrix factorization using Python, R and GenePattern Notebook implementations of CoGAPS

Parallel rapid expression and purification of proteins for crystallography (PREPX): large scale 1 L cultures

Abstract

Attachments

Steps

Expression

Cell lysis

Purification

Column regeneration: PD-10

Column regeneration: His GraviTrap

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