Escherichia coli-Based Production and Purification From Inclusion Bodies of Hetero-Oligomeric Mitochondrial Protease ClpXP in Active Form

Materials and reagents

Biological materials

1. E. coli Rosetta 2 (DE3) pLysS (Novagen, catalog number: 71403)

2. Encyclo Polymerase kit (Evrogen, catalog number: PK002S)

3. HS Taq DNA-polymerase (Evrogen, catalog number: PK017L)

4. Human cDNA generated by reverse transcription of RNA from HeLa cell line (BioinnLabs, catalog number: bn-hcdna)

5. Oligonucleotides (Table 1)

Table 1. Sequences of the used oligonucleotides

¹All oligonucleotides were synthesized in Evrogen, Moscow

5. QIAquick Gel Purification kit (Qiagen, catalog number: 28704)

6. QIAquick PCR Purification kit (Qiagen, catalog number: 28104)

7. QIAprep Spin Miniprep kit (Qiagen, catalog number: 28XXX)

8. Restriction endonuclease AgeI (NEB, catalog number: R0552S)

9. Restriction endonuclease NheI (NEB, catalog number: R0111S)

10. Restriction endonuclease XhoI (NEB, catalog number: R0146S)

11. T4 DNA ligase (Evrogen, catalog number: LK101S)

12. T7 Promoter (T7uni) primer (10 μM) (Evrogen, catalog number: PP006)

13. T7 Terminal primer (10 μM) (Evrogen, catalog number: PP007)

14. Vector plasmid: modified pET32b (Novagen, catalog number: 69016-3)

Reagents

1. β-casein from bovine milk (Bioultra, catalog number: C6905-250mg)

2. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250)

3. Acetic acid, glacial (Panreac, catalog number: 131008.1214)

4. Acrylamide (PanEco, catalog number: Х123)

5. Adenosine 5′-triphosphate (Sigma-Aldrich, catalog number: A-7699)

6. Agarose (Millipore, catalog number: 1.01236)

7. Ammonium acetate (Supelco, catalog number: 1.01116)

8. Ammonium persulfate (APS) (Bio–Rad, catalog number: 161-0700)

9. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518)

10. Bacto^TM yeast extract (Thermo Scientific, catalog number: 212750)

11. Bradford reagent (Sigma-Aldrich, catalog number: B6916)

12. Bromophenol blue (Sigma-Aldrich, catalog number: B0126)

13. Calcium chloride, dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: 21102)

14. Chloramphenicol (Sigma-Aldrich, catalog number: C0378)

15. Coomassie Brilliant Blue R-250 (Thermo Scientific, catalog number: 20278)

16. Difco^TM tryptone (Thermo Scientific, catalog number: 211921)

17. DNA 1 kb ladder (Evrogen, catalog number: NL001)

18. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)

19. Ethanol, 96% (SAFC, catalog number: 1.00971)

20. Ethidium bromide solution, 10 mg/mL (VWR International LCC, catalog number: X328-10ML)

21. Glycerol (Sigma-Aldrich, catalog number: G5516)

22. Glycine (Sigma-Aldrich, catalog number: G7126)

23. Hydrochloric acid (HCl) (Supelco, catalog number: 1.00317)

24. Imidazole (Acros Organics, catalog number: 12202-5000)

25. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I6758)

26. Magnesium chloride solution, 1 M (Sigma-Aldrich, catalog number: M1028)

27. Manganese chloride, dihydrate (MnCl₂·2H₂O) (Sigma-Aldrich, catalog number: 1.05934)

28. N,N’-methylenebisacrylamide (Bio-Rad, catalog number: 161-0201)

29. Ni-NTA agarose (Quigen, catalog number: 30210)

30. Prestained protein molecular weight marker, 20–120 kDa (Thermo Scientific, catalog number: 26612)

31. Sodium chloride (NaCl) (PanEco, catalog number: Х085/130315)

32. Sodium dodecyl sulfate (SDS) (Panreac, catalog number: 142363.1211)

33. Sodium hydroxide (Sigma-Aldrich, catalog number: 221465)

34. TEMED (Fluka, catalog number: 87690)

35. Tris acetate-EDTA buffer (Sigma-Aldrich, catalog number: T9650)

36. Tris(hydroxymethyl)aminomethane (Tris base) (Merck, catalog number: T1503)

37. Triton^TM X-100 (Bio-Rad, catalog number: 161-0407)

38. Urea (Sigma-Aldrich, catalog number: U5128)

39. Xylene cyanol (Sigma-Aldrich, catalog number: X4126)

40. Zinc sulfate heptahydrate (Sigma-Aldrich, catalog number: Z0251)

Solutions

1. 0.5 M EDTA pH 8.0 (see Recipes)

2. 0.5 M Tris-HCl, pH 6.8 (see Recipes)

3. 1 M IPTG (see Recipes)

4. 1.2% agarose gel (see Recipes)

5. 10% APS (see Recipes)

6. 10% SDS (see Recipes)

7. 10% (15%) SDS-PAGE (see Recipes)

8. 10% Triton X-100 solution (see Recipes)

9. 1 M Tris-HCl, pH 7.5 (see Recipes)

10. 1.5 M Tris-HCl, pH 8.8 (see Recipes)

11. 2 M imidazole, pH 7,5 (see Recipes)

12. 3 M NaCl (see Recipes)

13. 30% Acrylamide:Bis solution (see Recipes)

14. 4× SB buffer with β-Mercaptoethanol (see Recipes)

15. 5× SB buffer (see Recipes)

16. 5× SDS-PAGE running buffer (see Recipes)

17. 6× Loading buffer (Lb) (see Recipes)

18. 8 M urea (see Recipes)

19. 50× TAE Buffer (see Recipes)

20. Ampicillin (100 μg/mL) (see Recipes)

21. Assay buffer (AB) (see Recipes)

22. Bovine β-casein solution (10 μg/L) (see Recipes)

23. Buffer 1 (see Recipes)

24. Buffer 2 (see Recipes)

25. Buffer 3 (see Recipes)

26. Buffer 4 (see Recipes)

27. Buffer 5 (see Recipes)

28. Buffer 6 (see Recipes)

29. Buffer 7 (see Recipes)

30. Buffer 8 (see Recipes)

31. Buffer RF1 (see Recipes)

32. Buffer RF2 (see Recipes)

33. Chloramphenicol (25 μg/mL) (see Recipes)

34. Coomassie Brilliant Blue staining solution (see Recipes)

35. Fixing solution (see Recipes)

36. LB medium (see Recipes)

Recipes

Notes:

1. All chemicals should be handled according to the best laboratory practices and safety data sheets.

2. Unless otherwise specified, solutions should be filtered through a 0.22 μm membrane filter and stored at room temperature.

1. 0.5 M EDTA, pH 8.0

Dissolve 93.06 g of EDTA disodium salt dihydrate in approximately 400 mL of Milli-Q water, adjust the pH to 8.0 using NaOH (EDTA will not fully dissolve until the pH approaches 8.0), and adjust the final volume to 500 mL.

2. 0.5 M Tris-HCl, pH 6.8

Dissolve 30.3 g of Tris base in approximately 400 mL of Milli-Q water. While stirring, carefully adjust the pH to 6.8 using concentrated HCl. Once the pH is stable at 6.8 and the Tris base is completely dissolved, adjust the final volume to 500 mL.

3. 1 M IPTG

Dissolve 2.38 g of IPTG in approximately 8 mL of Milli-Q water. After complete dissolution, adjust the final volume to 10 mL. Sterilize the solution by filtration through a sterile 0.22 μm membrane filter. Aliquot into sterile tubes and store at –20 °C.

4. 1.2% agarose gel

Mix 0.6 g of agarose and 50 mL of 1× TAE buffer and melt until clear. Cool the mixture to 50–55 °C and add 2.5 µL of ethidium bromide solution. Mix thoroughly and pour into the mold.

Note: Ethidium bromide solution is toxic; wear gloves when handling.

5. 10% APS

Dissolve 1 g of ammonium persulfate in 10 mL of Milli-Q water. Mix thoroughly until completely dissolved. Aliquot into small volumes (e.g., 0.5–1 mL) and store at -20 °C. The solution is stable for at least 6 months when stored frozen. For best results in polymerization reactions, use freshly prepared solution or aliquots that have undergone less than five freeze-thaw cycles.

6. 10% SDS

Dissolve 50 g of SDS in approximately 400 mL of Milli-Q water with gentle stirring and heating to 50 °C to facilitate dissolution. Once fully dissolved, adjust the final volume to 500 mL.

Caution: Wear appropriate personal protective equipment, including a mask, when handling SDS powder to avoid inhalation.

7. 10% (15%) SDS-PAGE (for 2 mini-gels)

A. Resolving gel (10 mL)

Combine 3.3 (5) mL of 30% acrylamide/bis solution, 2.5 mL of 1.5 M Tris-Base (pH 8.8), 100 μL of 10% SDS, 4 (2.3) mL of Milli-Q water, and 100 μL of 10% APS in a 50 mL Falcon tube. Mix gently by swirling the tube. Immediately before pouring, add 10 μL of TEMED, mix gently but thoroughly, and pour the solution into the gel cassette using the 5 mL pipette. Leave appropriate space for the stacking gel and carefully overlay with isopropanol to ensure a flat interface. Allow gel to polymerize completely (approximately 15–30 min).

Caution: Acrylamide is a neurotoxin. Wear appropriate personal protective equipment when handling unpolymerized solutions.

B. 5% Stacking gel (2 mL)

After polymerization of the resolving gel, remove the overlay and rinse the gel surface with Milli-Q water. Combine 330 μL of 30% acrylamide/bis solution, 500 μL of 0.5 M Tris-Base (pH 6.8), 20 μL of 10% SDS, 1150 μL of Milli-Q water, and 20 μL of 10% APS in a Falcon tube. Mix gently by swirling the tube. Add 2 μL of TEMED, mix gently but thoroughly, and pour immediately on top of the polymerized resolving gel using the 1 mL pipette. Immediately insert a clean comb, avoiding air bubbles. Allow to polymerize completely (approximately 10–15 min).

8. 10% Triton X-100 solution

Add 10 mL of Triton X-100 to approximately 80 mL of Milli-Q water. Mix thoroughly using a magnetic stirrer until the solution becomes clear, and adjust the final volume to 100 mL. Store at 4 °C.

9. 1 M Tris-HCl, pH 7.5

Dissolve 121.14 g of Tris base in 900 mL of Milli-Q water with vigorous stirring. Once completely dissolved, adjust pH to 7.5 using concentrated HCl and adjust the final volume to 1 L.

10. 1.5 M Tris-HCl, pH 8.8

Dissolve 90.86 g of Tris base in approximately 400 mL of Milli-Q water. While stirring, adjust the pH to 8.8 using concentrated HCl. Once the pH is stable at 8.8 and the Tris base is completely dissolved, adjust the final volume to 500 mL.

11. 2 M imidazole, pH 7.5

Dissolve 136.2 g of imidazole in approximately 800 mL of Milli-Q water. While stirring, adjust the pH to 7.5 using concentrated HCl. Once the pH is stable and the imidazole is completely dissolved, adjust the final volume to 1 L.

12. 3 M NaCl

Dissolve 175.3 g of NaCl in approximately 800 mL of Milli-Q water with stirring. Once completely dissolved, adjust the final volume to 1 L.

13. 30% Acrylamide/Bis solution 29:1

Dissolve 145 g of acrylamide and 5 g of N,N’-methylenebisacrylamide in approximately 400 mL of Milli-Q water with gentle stirring. Once completely dissolved, adjust the final volume to 500 mL. Store the solution protected from light at 4 °C.

Caution: Acrylamide is a potent neurotoxin and a suspected carcinogen. Always wear appropriate personal protective equipment (gloves, lab coat, safety glasses) when handling unpolymerized acrylamide. Avoid inhalation of powder and skin contact. For safety reasons, the self-prepared solution can be replaced with commercially available reagents (e.g., Acrylamide/Bis Solution (29:1) 30%, Sigma-Aldrich, catalog number: A3574).

14. 4× SB buffer with β-mercaptoethanol

Combine 0.8 mL of 5× SB buffer and 0.2 mL of β-Mercaptoethanol. Mix thoroughly by pipetting. β-mercaptoethanol has a strong, unpleasant odor and is toxic; add it only in a fume hood.

15. 5× SB buffer

Transfer 3.125 mL of 0.5 M Tris-HCl (pH 6.8) to a clean 15 mL beaker. Add 5 mL of glycerol, 1.0 g of SDS, 5 mg of bromophenol blue, and distilled water to bring the total volume to 10 mL. Mix thoroughly until the SDS and dye are completely dissolved. Store at room temperature.

Note: SDS is an irritant; wear gloves when handling it and avoid inhaling dust.

16. 5× SDS-PAGE running buffer

Add approximately 800 mL of Milli-Q water to a 1 L graduated cylinder or beaker. Add 15 g of Tris base, 5 g of sodium dodecyl sulfate, and 72 g of glycine. Mix thoroughly using a magnetic stirrer until the solution becomes clear and homogeneous. Adjust the final volume to 1 L.

17. 6× Lb

Place 3 mL of glycerol in a 15 mL graduated tube. Add 25 mg of bromophenol blue, 25 mg of xylene cyanol, and distilled water to make up to 10 mL. Mix thoroughly until the dyes are completely dissolved. Store at 4 °C.

18. 8 M urea

Dissolve 480.45 g of urea in approximately 800 mL of Milli-Q water. Once the urea is completely dissolved, adjust the final volume to 1 L.

19. 50× TAE buffer

Dissolve 242 g of Tris base in approximately 700 mL of deionized water. Carefully add 57.1 mL of glacial acetic acid and 100 mL of 0.5 M EDTA, pH 8.0. Adjust the final volume to 1 L.

Caution: Prepare this solution in a well-ventilated area or fume hood due to the volatile and corrosive nature of glacial acetic acid. Wear appropriate personal protective equipment.

20. Ampicillin (100 mg/mL)

Dissolve 50 mg of ampicillin sodium salt in approximately 40 mL of sterile Milli-Q water. After complete dissolution, adjust the final volume to 50 mL. Sterilize the solution by filtration through a 0.22 μm membrane filter. Aliquot into sterile tubes and store at -20 °C.

21. Assay buffer (AB)

Combine 500 μL of 3 M NaCl, 500 μL of 1 M Tris-HCl, pH 7.5, 100 μL of 10% Triton X-100 solution, 50 μL of 1 M MgCl₂ solution, 25 μL of 10 mM ZnSO₄ solution, and 100 μL of 1 M ATP. Adjust the final volume to 10 mL with deionized water and mix briefly by vortexing. Due to the instability of ATP solutions, prepare the AB right before use, keep it refrigerated until use, and discard the remnants.

22. Bovine β-casein solution (10 μg/μL)

Weigh 10 mg of casein powder in a 1.5 mL Eppendorf tube. Add 900 μL of deionized water and 10 μL of 1 M Tris-HCl solution, pH 7.5. Mix gently and gradually increase the temperature to 80–85 °C for 10 min until you have a homogenous dispersion. Adjust the volume to 1 mL.

Critical: Do not boil the solution, as this may denature the protein. Cool to room temperature, aliquot, and store at -20 °C.

23. Buffer 1

Dissolve 480.45 g of urea in 950 mL of Milli-Q water, add 50 mL of 1 M Tris-HCl solution, pH 7.5, and mix thoroughly. Store at 4 °C.

24. Buffer 2

Dissolve 480.45 g of urea in 940 mL of Milli-Q water, add 50 mL of 1 M Tris-HCl solution, pH 7.5, and 10 mL of 2 M imidazole solution (pH 7.5), mix thoroughly, and store at 4 °C.

25. Buffer 3

Add 50 mL of 1 M Tris solution, pH 7.5, 50 mL of 3 M NaCl stock solution, and 12.5 mL of 2 M imidazole solution (pH 7.5) to 750 mL of 8 M urea solution. Adjust the final volume to 1 L with Milli-Q water. Mix thoroughly and store at 4 °C.

26. Buffer 4

Add 50 mL of 1 M Tris solution, pH 7.5, 50 mL of 3 M NaCl stock solution, and 15 mL of 2 M imidazole solution (pH 7.5) to 500 mL of 8 M urea solution. Adjust the final volume to 1 L with Milli-Q water. Mix thoroughly and store at 4 °C.

27. Buffer 5

Add 50 mL of 1 M Tris solution, pH 7.5, 50 mL of 3 M NaCl stock solution, and 17.5 mL of 2 M imidazole solution (pH 7.5) to 250 mL of 8 M urea solution. Adjust the final volume to 1 L with Milli-Q water. Mix thoroughly and store at 4 °C.

28. Buffer 6

Add 50 mL of 1 M Tris solution, pH 7.5, 50 mL of 3 M NaCl stock solution, and 20 mL of 2 M imidazole solution (pH 7.5) to 125 mL of 8 M urea solution. Adjust the final volume to 1 L with Milli-Q water. Mix thoroughly and store at 4 °C.

29. Buffer 7

Combine 50 mL of 1 M Tris solution, pH 7.5, 50 mL of 3 M NaCl stock solution, 25 mL of 2 M imidazole solution (pH 7.5), and 10 mL of 10% Triton X-100 solution. Adjust the final volume to 1 L with Milli-Q water. Mix thoroughly and store at 4 °C.

30. Buffer 8

Combine 50 mL of 1 M Tris solution, pH 7.5, 50 mL of 3 M NaCl stock solution, 200 mL of 2 M imidazole solution (pH 7.5), and 10 mL of 10% Triton X-100 solution. Adjust the final volume to 1 L with Milli-Q water. Mix thoroughly and store at 4 °C.

31. Buffer RF1

Dissolve 0.6 g of KCl, 0.075 g of CaCl₂·2H₂O, 0.5 g of MnCl₂·2H₂O, and 0.6 g of glycerol in approximately 800 mL of Milli-Q water. Mix thoroughly until all components are completely dissolved. Add 1.5 mL of CH₃COOH and mix thoroughly again. Adjust the final volume to 1 L. Sterilize the solution by filtration through a sterile 0.22 μm membrane filter. Aliquot into sterile tubes and store at 4 °C.

32. Buffer RF2

Dissolve 0.012 g of KCl, 0.11 g of CaCl₂·2H₂O, and 1.5 g of glycerol in approximately 800 mL of Milli-Q water. Mix thoroughly until all components are completely dissolved. Adjust the final volume to 1 L. Sterilize the solution by filtration through a sterile 0.22 μm membrane filter. Aliquot into sterile tubes and store at 4 °C.

33. Chloramphenicol (25 mg/mL)

Dissolve 12.5 mg of chloramphenicol in 50 mL of 96% ethanol. Mix thoroughly until completely dissolved. The solution is sterile and does not require filtration. Aliquot and store at -20 °C protected from light.

34. Coomassie Brilliant Blue staining solution

Dissolve 1.0 g of Coomassie Brilliant Blue R-250 in a mixture of 450 mL of 96% ethanol and 100 mL of glacial acetic acid until the dye is completely dissolved. Add 450 mL of Milli-Q water and mix thoroughly. Filter the solution through filter paper before use to remove any undissolved particles. Store in a tightly sealed container.

Caution: Glacial acetic acid is corrosive and volatile. Prepare this solution in a well-ventilated area or fume hood and wear appropriate personal protective equipment.

35. Fixing solution

Add 70 mL of glacial acetic acid to 800 mL of Milli-Q water in a 1 L graduated cylinder placed in a well-ventilated area or fume hood, mix thoroughly, and adjust the final volume to 1 L. Store in a tightly sealed container to prevent evaporation.

36. LB medium

Dissolve 10 g of Difco^TM tryptone, 5 g of Bacto^TM yeast extract, and 5 g of NaCl in 800 mL of Milli-Q water, mix thoroughly, and adjust the final volume to 1 L. Sterilize by autoclaving at 121 °C for 20 min.

Critical: The bacterial culture is inoculated into a medium cooled to room temperature. If necessary, the required antibiotics should be added immediately before inoculation.

Laboratory supplies

1. Falcon conical centrifuge tubes, 15 mL (Accumax, catalog number: Ac-ACT-15-R-S)

2. Falcon conical centrifuge tubes, 50 mL (Accumax, catalog number: Ac-ACT-50-S-W)

3. Centrifugal filters JetSpin, 2 mL 100 KDa MWCO (JetBioFil, catalog number: FTT100105)

4. Petri dishes (NEST, catalog number: N-752001)

5. Pipette dispensers (various volumes)

6. Pipette tips (various volumes)

7. Volumetric cylinders

8. Beakers

9. Erlenmeyer flasks

10. Round-bottomed flasks

11. Laboratory stands

12. Safe-lock tubes, 2.0 mL (SSI, catalog number: SSI-1310-00)

13. Safe-lock tubes, 0.5 mL (NEST, catalog number: N-605001)

14. Safe-lock tubes, 1.5 mL (SSI, catalog number: SSI-1260-00)

15. Safe-lock tubes, 0.2 mL (SSI, catalog number: SSI-3225-00)

Equipment

1. Autoclave VK-7501 (Tyumen medical, catalog number: 00003592)

2. Biological Safety Cabinet Labconco Purifier Logic+ Class II, Type A2 (Labconco, catalog number: 302481101)

3. ChemiDoc^TM MP Imaging System (Bio-Rad, catalog number: IBRD12003154)

4. CLARIOstar^® Plus multi-mode plate reader (BMG Labtech, catalog number: 430-501S-F)

5. Electrophoresis Power Supply Bio-Rad PowerPac Basic (Bio-Rad, catalog number: 1645050)

6. Glass vacuum filtration device (Whatman, catalog number: WHA10441001)

7. High-Speed benchtop centrifuge Eppendorf Centrifuge 5804R (Eppendorf, catalog number: 5805000010)

8. Horizontal Electrophoresis Unit (Labotronics, catalog number: LB-10HEU)

9. Lab Balance Mettler Toledo XPE204 (Mettler Toledo, catalog number: 30087643)

10. Lab freezer (-20 °C)

11. Lab refrigerator (2–8°C)

12. Magnetic Stirrer Heidolph MR Hei-Standard (Heidolph, catalog number: 505-20000-00)

13. Milli-Q Direct Water Purification System (Millipore, catalog number: ZR0Q008US)

14. Multiskan FC with thermostat (Thermo Fisher Scientific, catalog number: 51119100)

15. New Brunswick Innova^® 44/44R Stackable Incubator Shaker (Eppendorf, catalog number: M1282-0002)

16. pH-meter Mettler Toledo S20 SevenEasy^TM (Mettler Toledo, catalog number: S20)

17. Solid-state thermostat TT-1 Gnome (DNA Technology, catalog number: 00010014049)

18. Standard Incubator Binder BD 56 (Binder, catalog number: 9010-0323)

19. Heated Lid Thermal Cycler (MJ Research, catalog number: PTC-100)

20. Ultra-low (-70 °C) temperature freezer Sanyo MDF-U53V (Sanyo, catalog number: SM9910073)

21. Ultrasonic disintegrator (Ningbo Scientz Biotechnology Co., China, model: JY92-IIN)

22. Vertical Polyacrylamide Gel Electrophoresis System Bio-Rad Mini-PROTEAN Tetra Cell (Bio-Rad, catalog number: 1658005EDU)

Software and datasets

1. Unipro UGENE 53.1 [27–29], to analyze sequences and design oligonucleotides

2. Image J [30], for densitometry analysis

Procedure

A. Production of plasmid constructs and producing clones for prokaryotic expression of ClpXP protease

Note: All bacterial cultivation operations are carried out under sterile conditions. Unless otherwise specified, all overnight cultures are incubated for 16 h (overnight) at 37 °C and 200 rpm on an orbital thermoshaker.

1. Amplify the coding regions of the ClpX and ClpP proteases using polymerase chain reaction (PCR) on human cDNA using the oligonucleotides listed in Table 1 and Encyclo polymerase. Introduce restriction sites for NheI, AgeI, and XhoI at the 5′ ends of the oligonucleotides. Select PCR conditions according to the Encyclo polymerase manufacturer’s recommendations. The thermal cycling includes initial denaturation at 94 °C for 3 min, 25 cycles of [denaturation at 95 °C for 25 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min (ClpP) and 2 min (ClpX)], and final extension at -72 °C for 5 min.

2. Validate PCR products using agarose gel electrophoresis (see section A1 below) and purify using the QIAquick PCR Purification Kit according to the manufacturer’s recommendations.

3. Digest purified PCR products and vector plasmid pET32b with the appropriate restriction endonucleases, with consequent purification of the restriction products from agarose gel using the QIAquick Gel Purification kit, according to the manufacturer’s recommendations.

4. Incubate the ligation mixtures (prepared according to the T4 DNA-ligase manufacturer’s recommendations) overnight (16 h) at 10 °C. After incubation, use the ligation mixtures to transform competent E. coli cells, strain MH-1 (see section A2). Plate transformed cells on LB Petri dishes containing 100 μg/mL ampicillin.

5. Select individual colonies for plasmid DNA isolation by the sizes of the inserts using PCR with T7-universal primers and HS Taq DNA-polymerase.

6. Isolate plasmid DNAs from 3 mL of overnight cultures of positive (insert-containing) clones using the QIAprep Spin Miniprep kit, according to the manufacturer’s recommendations.

7. Test purified plasmid DNAs for the absence of cloning-related mutations using automated sequencing. In our work, Sanger sequencing was performed on an Applied Biosystems 3500xL genetic analyzer (Evrogen, Moscow).

8. After successful validation, transform sequenced plasmids into E. coli cells, strain Rosetta 2 (DE3) pLysS (see section A2). Plate transformed cells on LB Petri dishes containing 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. Use single colonies for expression of ClpX and ClpP proteases.

A1. Agarose gel electrophoresis

1. Attach a comb to an electrophoresis tray, then pour a layer of 1.2% agarose in 1× TAE buffer (containing 0.5 μg/mL ethidium bromide) into the tray and allow the gel to solidify.

2. Load 10 μL of loading DNA samples (prepared by combining 8 μL of DNA sample and 2 μL of 6× Lb) into the wells of the gel. Perform the electrophoresis in 1× TAE at 5 V/cm.

A2. Preparation of chemically competent E. coli cells and plasmid DNA transformation

1. Dilute the overnight culture grown in LB medium 100-fold and grow the cells to OD600 = 0.4–0.5 at 37 °C on an orbital thermoshaker (180 rpm).

2. Transfer the cells to ice and pellet in a centrifuge at 4,000× g for 5 min.

3. Discard the supernatant, resuspend the pellet in cold RF1 buffer (3.5 mL per 10 mL of culture), and incubate the resuspended pellet on ice for 15 min.

4. Pellet the cells as described in step A2.2.

5. Discard the supernatant, resuspend the pellet in cold RF2 buffer (0.8 mL per 10 mL of culture), and incubate the resuspended pellet on ice for 15 min.

6. Prepare100 µL aliquots of cells and store them at -70 °C.

7. For transformation, add 100 ng of expression plasmid to ice-thawed competent cells and incubate the mixture for 60 min on ice.

8. Then, heat shock the cells at 42 °C for 2 min and incubate them on ice for 2 min.

9. Next, add 500 μL of LB medium, incubate at 37 °C for 60 min without shaking, and plate the transformants on Petri dishes with solid LB medium containing the indicated antibiotic(s) with consequent overnight (16 h) incubation at 37 °C.

B. Inducible expression of recombinant proteins

Note: For any particular protein, the optimal expression strain and conditions (e.g., induction temperature and inducer concentration) may vary. Therefore, before proceeding with this protocol, users should perform their own small-scale expression tests to identify the conditions that maximize the level of the target protein expression. In our case, the E. coli strain Rosetta 2 (DE3) pLysS was used for the expression of both ClpX and ClpP proteases, while the optimal induction temperature was 24 and 37 °C for ClpX and ClpP, respectively.

1. Inoculate a single well-formed colony into a 50-mL Falcon tube containing 5–10 mL of LB medium with 25 μg/mL chloramphenicol and 100 μg/mL ampicillin and incubate for 16 h (overnight) at 37 °C with 200 rpm rotation speed on the orbital thermoshaker.

2. Next morning, dilute the overnight cultures of ClpP and ClpX 50-fold in LB medium containing 100 μg/mL ampicillin and incubate for 2 h at 37 °C to an OD600 of 0.4–0.6.

3. Then, induce expression by adding IPTG to a concentration of 1 mM. Culture the ClpP-expressing cells at 37 °C for 5 h, and culture the ClpX-expressing cells at 25 °C for 16 h.

4. Collect the cells by centrifugation at 4,000× g for 10 min. Freeze and store the collected cells at -20 °C for subsequent protein isolation.

C. Cell lysis and inclusion body isolation

Note: Unless otherwise specified, all procedures should be performed at 4 °C (on ice or in a cold room). Use pre-cooled buffers and centrifuge rotors. The sonication should be performed in a beaker placed on an ice bath until the supernatant becomes clear.

1. Harvest the cells from the 400 mL culture (200 mL of ClpP and 200 mL of ClpX) by centrifugation at 4,000× g for 15 min. Carefully decant and discard the supernatant. Either immediately use the cell pellet for purification or freeze it at -20 or -80 °C for long-term storage.

2. Completely resuspend the cell pellet in 35 mL of Buffer 1 by short-time vortexing to keep the lysate cool.

3. Disintegrate the cellular membranes and DNA using an ultrasonic disintegrator with the following settings to minimize heating: Tip 6 mm, amplitude 100%; 10 s pulse-on, 10 s pulse-off; for a total time of 600 s.

4. Transfer the lysate into an appropriate centrifuge tube and centrifuge it at 10,000× g for 20 min at 4 °C to pellet the urea-insoluble fraction.

5. For protein isolation, use the supernatant.

D. Resin preparation, affinity chromatography, and on-column refolding

Notes:

1. Prepare the resin at room temperature just before the protein purification. Either the batch method or the gravity column method can be used for equilibration. The prepacked column can be stored at 4 °C with the end caps closed (with a preservative, e.g., 20% ethanol, for long-term storage).

2. In this particular case of the ClpXP purification from 400 mL of cell culture, we used 1 mL of Ni-NTA resin, the batch method for protein binding, and a self-made gravity column for washing and elution. Washing conditions (e.g., proposed step gradient of urea and imidazole) were optimal to eliminate unspecific binding without losing the target protein. Buffers 7 and 8 contained Triton X-100 to prevent protein aggregation. However, the optimal chromatography conditions may differ for other proteins and other types of resins used. Therefore, users should conduct their own small-scale protein purification experiments to find optimal conditions.

3. All protein fractions collected during purification were analyzed by SDS-PAGE.

4. We recommend performing the purification in a refrigerated cabinet, cold room, or, at least, using pre-cooled buffers.

5. The resin can be regenerated as recommended by the manufacturer.

6. If protein isolation needs to be scaled up or if procedure automation is desired, any FPLC chromatograph can be used. The protein can be loaded onto the resin in a single step, and the step gradient can be replaced with a linear gradient. It is recommended to perform all manipulations in a cold room or using prechilled buffers.

1. Gently mix the bottle with Ni-NTA resin, place 2 mL of Ni-NTA resin suspension (50% slurry in 20% EtOH) in a 2 mL tube, and centrifuge at 8,000× g for 30 s. Discard the supernatant. Wash the resin with 1 volume of water and then with 2 volumes of Buffer 1.

2. Adjust the volume of the clarified lysate to 49 mL with Buffer 1 and add imidazole to a concentration of 20 mM (per 50 mL).

Note: Line 2 in Figure 1A illustrates the 8 M urea-soluble protein fraction loaded onto Ni-NTA resin.

3. Add 1 mL of the equilibrated Ni-NTA resin to the protein lysate and incubate the mixture overnight at 8 °C with permanent rotation, providing optimal conditions for protein binding.

4. After incubation, transfer the suspension to the empty column and leave the column with its end caps open for removing the flowthrough fraction by gravity.

Note: Line 4 in Figure 1A illustrates the flowthrough fraction from the Ni-NTA resin.

5. To eliminate the unspecific binding and refold the denatured ClpXP subunits, perform stepwise decreasing the urea concentration from 8 to 0 M and increasing the imidazole from 20 to 50 mM. To do this, consequently wash the resin with 5 volumes of Buffers 2–7 by gravity.

Note: Line 5 in Figure 1A illustrates the combined washing fraction.

6. After the washing/refolding step, perform the elution by gravity with Buffer 8. Collect 1–2 mL fractions manually. Monitor the elution efficiency using the Bradford reagent according to the manufacturer’s recommendations.

7. Pool together the ClpXP-containing fractions and concentrate the protein solution to 2–5 mg/mL using a centrifuge concentrator with a 100 kDa cutoff.

Note: Lines 6 and 8 in Figure 1A illustrate the purified ClpXP after the concentration step, while lines 7 and 9 illustrate the flowthrough from the 100 kDa concentrator.

8. Since ClpXP was a protease, in order to avoid autoproteolysis, add glycerol to a final concentration of 50% and keep the resulting protein solution at -20 °C.

E. Purity analysis by SDS-PAGE

1. Prepare the protein samples (e.g., 10–20 μg of protein or 10–15 µL of fractions) by mixing them 3:1 by volume with 4× SB and denaturing at 90 °C for 3 min.

2. Load the samples into wells of a 10% or 15% polyacrylamide gel.

3. Perform the electrophoresis in a chamber filled with electrophoresis buffer at a constant voltage of 120 V until the dye front reaches the bottom of the gel.

4. Place the gel in fixing solution for 5–10 min.

5. Transfer the gel to hot (50–60 °C) Coomassie Brilliant Blue staining solution for 10–20 min.

6. Remove the excess dye by rinsing the gel with hot fixing solution (50–60 °C) until clear protein bands appear on a transparent background.

Data analysis

A. Protein purity assessment

1. All protein fractions (protein lysate, flowthrough, column wash, and elution fraction) were analyzed by SDS-PAGE using 10% polyacrylamide gels (Figure 1).

2. Protein bands were visualized by Coomassie Brilliant Blue R-250 staining (Figure 1A).

3. Protein purity was confirmed by densitometric analysis of gel images using ImageJ [28]. As shown in Figure 1B and C, approximately 63.8% and 74.1% of the total protein was attributed to the ClpX subunits in elution fractions 1 and 2, respectively, while 27.3% and 20.9% of the total protein was attributed to the ClpP subunits in elution fractions 1 and 2, respectively. That indicates that the purity of the purified ClpXP complexes was approximately 91.1% and 95% in elution fractions 1 and 2, respectively. In both cases, satisfactory protein purity (greater than 90%) was achieved.

B. Assessment of protein yield and formation of high-molecular-weight complex

1. Protein concentration was determined using Bradford reagent according to the manufacturer’s recommendations, with bovine serum albumin as the standard.

2. Total protein yield was calculated on the basis of protein concentration in elution fractions. 6.75 mg of ClpXP protease complex was obtained from 400 mL of E. coli culture (200 mL of ClpP-expressed cells and 200 mL of ClpX-expressed cells).

3. SDS-PAGE analysis confirmed the absence of the individual subunits of the ClpXP complex in the flowthrough fraction from the centrifuge concentrator device with a 100 kDa cutoff (Figure 1A: line 7 vs. 6, and line 9 vs. 8). That indicates successful formation of the high-molecular-weight complex.

Figure 1. Purification of ClpXP and analysis of the collected protein fractions. (A) 10% SDS-PAGE analysis of the ClpXP purification: 1, prestained protein ladder; 2, 8 M urea-soluble fraction; 3, 8 M urea-unsoluble fraction; 4, flowthrough fraction; 5, wash fraction; 6 and 8, elution fractions 1 and 2, respectively, concentrated with a centrifuge concentrator with a 100 kDa cutoff; 7 and 9, flowthrough fractions from a centrifuge concentrator with a 100 kDa cutoff obtained after concentrating elution fractions 1 and 2, respectively; 10, prestained protein ladder. ClpX and ClpP positions are indicated by arrows at the left. (B, C) Purity assessment of elution fractions 1 and 2 by densitometric analysis using ImageJ software.

C. Assessment of the desirable stoichiometry of the ClpXP complex

Using the above protocol, high-molecular-weight mitochondrial protease complex ClpXP was obtained after separate expression in E. coli of two His-tagged subunits and their combined purification from inclusion bodies using Ni-NTA IMAC in denatured conditions with consequent on-column refolding. Separate expression of the two subunits allowed individual optimization of the temperature conditions that provided the maximum yield of the final complex.

Figure 2A shows that combined protein purification after expression of both ClpP and ClpX at the same temperature (either 24 or 37 °C) yielded undesirable stoichiometry of the subunits of the ClpXP complex and a higher level of contaminants, whereas Figure 2B shows that combined protein isolation after their individual expression at different induction times and temperatures (5 h of induction at 37 °C for ClpP and 16 h of induction at 24 °C for ClpX) yielded the optimal combination of complex stoichiometry and purity.

Figure 2. Analysis of the ClpXP complex purification upon expression of the two subunits using similar or different conditions. (A) Expression of both subunits either at 24 °C (left) or 37 °C (right). (B) Expression of the ClpX and ClpP subunits at their optimal expression conditions: for ClpP, it was 5 h of induction at 37 °C; for ClpX, it was 16 h of induction at 24 °C. M, prestained protein ladder; 1, flowthrough fraction; 2, combined wash fraction; 3, elution fraction. ClpX and ClpP positions are indicated by arrows at the left.

D. Quality control criteria

1. The purity of the obtained ClpXP fraction, determined by densitometry, should be over 90% of total protein.

2. Protein fraction with significant proteolytic degradation or smearing on SDS-PAGE should be excluded.

3. The obtained protein must have functional activity.

Validation of protocol

Final validation of the presented protocol required assessment of the functional activity of the human ClpXP protease complex obtained in the heterologous expression system. The proteolytic activity of the purified ClpXP complex was analyzed by its ability to digest casein. Casein is widely used as the standard model substrate for evaluating mitochondrial proteases (such as Lon and ClpP). Since mitochondrial matrix proteases function primarily as quality-control enzymes, casein, as an intrinsically disordered protein, structurally mirrors a denatured or unfolded mitochondrial protein [31]. Protocol validation was performed using three independent protein preparations (biological replicates). Figure 3A illustrates the typical pattern of beta-casein hydrolysis by the purified ClpXP complex. Figure 3B shows the mean percentage of casein hydrolysis by ClpXP across three independent experiments.

A. Assessment of the proteolytic activity of the purified ClpXP complex

1. All points of the assay were done in triplicate.

2. The assay was carried out in 0.5 mL tubes; the assay volume was 30 μL.

3. The assay buffer (AB) was prepared and well mixed as described in Recipes.

4. The required volume of 10 μg/μL bovine β-casein solution (2 μg per reaction) was added to the appropriate AB volume. AB volume was calculated as follows: (30 μL × N of assays) × 1.1. The β-casein volume was calculated as follows: (0.2 μL × N of assays) × 1.1. The mixture was mixed by pipetting and aliquoted at 30 μL into 0.5 mL tubes in accordance with the number of reactions.

5. Enzyme serial dilutions in AB were added to the reaction replicates (the maximum added volume should not exceed 3 μL), mixed, spun briefly, and incubated at 37 °C for 3 h.

6. 15 μL of 4× SB were added to the hydrolysis samples. The samples were incubated at 90 °C for 5 min (to halt hydrolysis) and spun briefly. The samples were loaded by 20 μL into the wells of 15% SDS-PAAGE and resolved by electrophoresis. The gel was stained with Coomassie Brilliant Blue (see Figure 3A).

7. The stained gels were digitized using a Bio-Rad gel documentation system, and the casein bands were quantified via densitometric analysis using ImageJ software.

Figure 3A shows SDS-PAGE analysis of ClpXP-dependent hydrolysis of 2 µg of bovine β-casein. In the left line, where the maximum amount of enzyme (50 ng) was used, all casein was digested. In subsequent lines, the hydrolysis efficiency decreases in accordance with the decreasing amounts of ClpXP. The right line shows unhydrolyzed casein in the absence of the protease complex. Figure 3B shows statistical analysis of the three independent purification/hydrolysis experiments.

Figure 3. Analysis of ClpXP-dependent hydrolysis of β-casein. (A) 15% SDS-PAGE analysis of the bovine β-casein (2 μg) hydrolysis by the purified ClpXP complex: the ClpXP amount is indicated on the bottom; ClpX, ClpP, and casein positions are indicated by arrows at the right. M, prestained protein ladder. (B) Densitometric analysis of β-casein bands using the ImageJ software. Data are presented as the mean percentage of the remaining (unhydrolyzed) β-casein, where the initial load (2 μg) is taken as 100%, ± SD (n = 3, i.e. ClpXP was produced in three independent expression experiments).

General notes and troubleshooting

General notes

1. Protein storage:

a. Refolded protein should be stored at –20 °C into 50% glycerol.

b. Optimal protein concentration is 1–2.5 mg/mL.

2. Buffer and gradient optimization:

a. The chaotropic agent for the dilution of the inclusion bodies can be optimized (8 M urea or 6 M guanidine hydrochloride).

b. The stepwise gradient for decreasing urea concentration from 8 to 0 M and increasing imidazole from 20 to 50 mM can be optimized.

c. Ionic strength and pH conditions in the buffer solutions should be adapted to the nature of the protein purified.

d. For proteins requiring specific redox agents, such as β-mercaptoethanol or DTT, these agents should be included in the buffer solutions.

e. Different mild detergents or their combination could be tested.

3. System applicability: In case of different affinity tags (GST, MBP), inclusion bodies should be washed, solubilized, and refolded before the affinity purification step.

4. Scale considerations:

a. When the production volume is increased, the volumes of the resin and buffer solutions should be scaled up proportionally.

b. For larger scales, automation is recommended: any FPLC chromatograph with appropriate columns can be used.

5. Quality control:

a. All protein fractions (protein lysate, flowthrough, column wash, and elution fraction) should be analyzed by SDS-PAGE using appropriate percentage polyacrylamide gels.

b. Freshly prepared buffer solution, regular cleaning of the working area, and temperature control during the purification process are strongly recommended.

Troubleshooting

Acknowledgments

The work was carried out with the financial support of the Russian Science Foundation project 24–74-10107.

Author contributions

Conceptualization, E.V.S.; Investigation, O.S.E., E.V.S., T.V.R.; Writing—Original Draft, O.S.E., E.V.S., T.V.R.; Writing—Review & Editing, A.A.K., A.A.B.; Funding acquisition, A.A.K., A.A.B.

Competing interests

The authors declare that they have no competing interests.

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