Escherichia coli-Based Production and Purification From Inclusion Bodies of Hetero-Oligomeric Mitochondrial Protease ClpXP in Active Form
发布: 2026年11月20日第16卷第22期 DOI: 10.21769/BioProtoc.5865 浏览次数: 14
评审: Alejandro Avilés-ReyesAnonymous reviewer(s)
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Q&A
Abstract
Obtaining a functionally active mitochondrial protease complex ClpXP using the Escherichia coli–based expression system is complicated by two factors: (i) complex hetero-oligomeric structure of this multi-subunit complex requires correct assembly of the subunits in heterological environment, while (ii) restoration of the catalytic activity during the expression is toxic for the host cells. This paper presents a simple and low-cost approach that combines separate expression of two ClpXP subunits in insoluble form, their combined purification from inclusion bodies by Ni-NTA metal affinity chromatography, and refolding of the active hetero-oligomeric protein directly on the column by stepwise decreasing the urea concentration. Separate expression allows individual optimization of expressional conditions. The insoluble state of expressed subunits prevents host toxicity and increases the yield. The combined denaturation and immobilization on the column promote the formation of a native hetero-oligomeric structure during refolding, enabling the isolation of the enzyme in its active form. Elution with imidazole ensures the release of correctly folded protein immediately after the removal of denaturing stress, minimizing the time the protease remains in a partially denatured state. The proposed protocol is reproducible and can be applied to the production of other hetero-oligomeric recombinant proteases in active forms.
Key features
• Isolation of active ClpXP complexes by separately expressing insoluble ClpX and ClpP subunits in E. coli, then purifying them together via gravity chromatography.
• Individually expressed subunits are purified and refolded on a Ni-NTA column using urea and imidazole gradients, sequentially removing the denaturant via competitive elution.
• Controlled removal of denaturing stress on the solid phase prevents subunit aggregation and promotes native hetero-oligomeric formation, yielding a catalytically active enzyme.
• Ni-NTA sorbent with urea-imidazole gradient ensures highly selective isolation of the multimeric complex with confirmed activity, indicating the method’s suitability for purification of hetero-oligomeric proteases.
Keywords: Recombinant hetero-oligomeric protein
Inclusion body
Refolding
Immobilized metal affinity chromatography (IMAC)
Purification of multimeric protein in active form
Background
Heterologous expression of recombinant proteins is a fundamental technique in modern biotechnology. However, its efficiency is significantly limited by the mismatch between the molecular apparatus of the host cell and the structural and functional requirements of the target product [1,2]. This problem is most acute in the production of complex proteins characterized by a multidomain organization, the presence of multiple disulfide bonds, cofactor-dependent regions, or an oligomeric quaternary structure [3,4]. Such molecules are evolutionarily adapted to the specific microenvironment of donor cells, and their reproduction in heterologous systems is associated with fundamental biophysical barriers.
A key limitation of prokaryotic systems, particularly Escherichia coli, remains the inability to ensure the correct folding of complex polypeptides. The intracellular environment of bacteria is characterized by a reducing potential that prevents the formation of stable disulfide bonds, as well as the absence of specialized chaperone systems necessary for the assembly of multisubunit complexes [5,6], resulting in the accumulation of the target protein within inclusion bodies—insoluble aggregates consisting of a mixture of misfolded conformers [7]. Proteins containing prosthetic groups (heme, iron-sulfur clusters, flavin cofactors) pose an additional challenge, as their functional activity directly depends not only on the correct folding of the polypeptide chain but also on the correct incorporation of the cofactor, which is not always achieved in heterologous systems [8,9].
At the same time, the formation of inclusion bodies rescues host cells from the toxicity of heterologous proteins [10]. For example, most proteases become inherently dangerous when expressed in E. coli cells due to their ability to hydrolyze host cell proteins, while their autoproteolytic capacity reduces the production level [11]. In this case, the production of recombinant proteases in the form of inactive inclusion bodies followed by isolation under denaturing conditions is an effective isolation strategy provided that there are refolding conditions that restore the native protein structure [12].
Removal of target proteins from inclusion bodies requires the development of individual refolding protocols for each molecular target. Methods based on dilution or dialysis are characterized by low reproducibility when working with multisubunit protein complexes due to their high tendency to aggregate during intermediate stages of denaturant removal [13]. Chromatographic strategies that allow immobilization of the target protein on a solid phase and controlled removal of denaturing stress are considered more promising. This is particularly critical for oligomeric complexes, where spatial separation of molecules minimizes nonspecific association [14]. However, even under optimal refolding conditions, restoration of the native conformation for proteins with complex architecture remains a process difficult to predict, requiring empirical selection of parameters for each specific target.
Human mitochondrial protease complex ClpXP consists of two subunits: ClpX, an ATP-dependent chaperone that recognizes protein substrates and unfolds them using energy derived from ATP hydrolysis (UniProtKB O76031), and proteolytic subunit ClpP (UniProtKB Q16740) [15–17]. ClpXP is a key player in the mitochondrial proteostasis system, being involved in the maintenance of cellular energy supply; dysfunction of this complex is associated with a wide range of pathologies [18]. The study of human ClpXP is of particular importance, not only because of its biomedical significance but also because of the unique structural and regulatory organization of the complex, as revealed in recent cryo-electron studies [19]. ClpX forms a homohexameric ring with axial processing, and ClpP forms a tetradecameric structure of two heptameric rings within which 14 catalytic centers are located [20,21]. Expression of the upper components in E. coli leads to the accumulation of the target protein in inclusion bodies, and traditional refolding methods (dialysis, dilution) do not lead to the correct assembly of the multimeric structure [22]. A fundamental breakthrough in ClpXP purification was achieved when the use of a catalytically inactive mutant of ClpP (ClpP S97A in E. coli or S98A in S. aureus), called ClpPtrap, was proposed [22–25]. ClpPtrap retained the ability to bind substrates but was unable to cleave them, allowing co-purification of the ClpXP complex together with the captured protein substrates. At the same time, the E. coli extract-based cell-free expression system was used for successfully obtaining the active ClpXP protease complex [26].
Here, we report a novel, efficient method for obtaining functionally active ClpXP protease. The method is based on the separate expression of ClpX and ClpP in Escherichia coli in an insoluble state and their united purification in denaturing conditions with consequent refolding. The purification protocol includes immobilized metal affinity chromatography (IMAC) on the Ni-NTA gravity column, during which the stepwise increasing imidazole concentration (for removing unspecific bound proteins) was combined with stepwise decreasing urea concentration (refolding). Controlled removal of denaturing stress on the solid phase prevents nonspecific subunit aggregation and facilitates the formation of a native hetero-oligomeric structure, enabling the isolation of the enzyme in a catalytically active conformation. Key advantages of this approach include the integration of solubilization, affinity purification, and renaturation in a single cycle, which provides high selective isolation of the multimeric complex and reproducibility of the results. The presented strategy can be adapted to produce other hetero-oligomeric recombinant proteases.
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
| Name | Sequence1, 5′→3′ |
| CLPP AgeI fr | caccaCCGGTATGggaTGGCCCGGAATATTGGTAGG |
| CLPP XhoI rev | ccacctcgagCTAGGTGCTAGCTGGGACAGGTTCTGC |
| CLPX AgeI fr | caccACCGGTATGggaCCCAGCTGCGGTGCTTGTACTTG |
| CLPX NheI rev | ccacGCTAGCCTAGCTGTTTGCAGCATCTGCTTGGC |
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. BactoTM 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. DifcoTM 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. TritonTM 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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| EDTA disodium salt dihydrate | 0.5 M | 93.06 g |
| Milli-Q water | n/a | to 500 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 0.5 M | 30.3 g |
| Milli-Q water | n/a | to 500 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| IPTG | 1 M | 2.38 g |
| Milli-Q water | n/a | to 10 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1× TAE | 1× | 50 mL |
| Ethidium bromide | 0.5 μg/mL | 2.5 μL |
| Agarose | 1.2% | 0.6 g |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Ammonium persulfate | 10% (w/v) | 1 g |
| Milli-Q water | n/a | 10 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Sodium dodecyl sulfate | 10% (w/v) | 50 g |
| Milli-Q water | n/a | to 500 mL |
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)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 30% Acrylamide/Bis solution | 10% (15%) | 3.3 (5) mL |
| 1.5 M Tris-HCl, pH 8.8 | 0.375 M | 2.5 mL |
| 10% SDS | 0.1% | 100 μL |
| 10% APS | 0.1% | 100 μL |
| TEMED | 0.01% | 10 μL |
| Milli-Q water | n/a | 4 (2.3) 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)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 30% Acrylamide/Bis solution | 5% | 330 μL |
| 0.5 M Tris-HCl, pH 6.8 | 0.125 M | 500 μL |
| 10% SDS | 0.1% | 20 μL |
| 10% APS | 0.1% | 20 μL |
| TEMED | 0.01% | 2 μL |
| Milli-Q water | n/a | 1150 μL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Triton X-100 | 10% (v/v) | 10 mL |
| Milli-Q water | n/a | to 100 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 1 M | 121.14 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 1.5 M | 90.86 g |
| Milli-Q water | n/a | to 500 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Imidazole | 2 M | 136.2 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| NaCl | 3 M | 175.3 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Acrylamide | 29% (w/v) | 145 g |
| N,N’-methylenebisacrylamide | 1% (w/v) | 5 g |
| Milli-Q water | n/a | to 500 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 5× SB buffer | 4× | 0.8 mL |
| β-Mercaptoethanol | 20% (2.86 M) | 0.2 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 0.5 М Tris-HCl, pH 6.8 | 312.5 mМ | 3.125 mL |
| SDS | 10% | 1.0 g |
| Glycerol | 50% | 5 mL |
| Bromophenol blue | 0.05% | 5 mg |
| Milli-Q water | n/a | To 10 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 0.125 M | 15 g |
| Sodium dodecyl sulfate | 1.0% | 5 g |
| Glycine | 0.96 M | 72 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Glycerol | 30% | 3 mL |
| Bromophenol blue | 0.25% | 25 mg |
| Xylene cyanol | 0.25% | 25 mg |
| Milli-Q water | n/a | To 10 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Urea | 8 M | 480.45 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 2 M | 242 g |
| Glacial acetic acid | 5.7% (v/v) | 57.1 mL |
| 0.5 M EDTA, pH 8.0 | 0.05 M | 100 mL |
| Milli-Q water | n/a | to 1 L |
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)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Ampicillin sodium salt | 100 mg/mL | 50 mg |
| Sterile Milli-Q water | n/a | to 50 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)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 3M NaCl | 150 mM | 500 μL |
| 1 M Tris-HCl, pH 7.5 | 50 mM | 500 μL |
| 10% Triton X-100 solution | 0.1% | 100 μL |
| 1 M MgCl2 | 5 mM | 50 μL |
| 10 mM ZnSO4 | 25 μM | 25 μL |
| Milli-Q water | n/a | to 10 mL |
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)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Casein powder | 10 mg/mL | 10 mg |
| 1 M Tris-HCl, pH 7.5 | 10 mM | 10 μL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| Urea | 8 M | 480.45 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 47.1 mM | 50 mL |
| 2 M imidazole | 20 mM | 10 mL |
| 8 M urea | 7.5 M | 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| 3 M NaCl | 150 mM | 50 mL |
| 8 M urea | 6 M | 750 mL |
| 2 M imidazole | 25 mM | 12.5 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| 3 M NaCl | 150 mM | 50 mL |
| 8 M urea | 4 M | 500 mL |
| 2 M imidazole | 30 mM | 15 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| 3 M NaCl | 150 mM | 50 mL |
| 8 M urea | 2 M | 250 mL |
| 2 M imidazole | 35 mM | 17.5 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| 3 M NaCl | 150 mM | 50 mL |
| 8 M urea | 1 M | 125 mL |
| 2 M imidazole | 40 mM | 20 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| 3 M NaCl | 150 mM | 50 mL |
| 2 M imidazole | 50 mM | 25 mL |
| 10% Triton X-100 | 0.1% | 10 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 1 M Tris-HCl, pH 7.5 | 50 mM | 50 mL |
| 3 M NaCl | 150 mM | 50 mL |
| 2 M imidazole | 400 mM | 200 mL |
| 10% Triton X-100 | 0.1% Triton | 10 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| KCl | 4.9 mM | 0.6 g |
| CH3COOH 1 M | 1.5 mM | 1.5 mL |
| CaCl2·2H2O | 0.51 mM | 0.075 g |
| MnCl2·2H2O | 3.1 mM | 0.5 g |
| Glycerol | 6.52 mM | 0.6 g |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| KCl | 0.1 mM | 0.012 g |
| CaCl2·2H2O | 0.748 mM | 0.11 g |
| Glycerol | 16.3 mM | 1.5 g |
| Milli-Q water | n/a | to 1 L |
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)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Chloramphenicol | 25 mg/mL | 12.5 mg |
| Ethanol | 96% (v/v) | 50 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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Coomassie Brilliant Blue R-250 | 0.1% (w/v) | 1.0 g |
| Ethanol, 96% | 43.2% (v/v) | 450 mL |
| Glacial acetic acid | 10% (v/v) | 100 mL |
| Milli-Q water | n/a | 450 mL |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Glacial acetic acid | 7% (v/v) | 70 mL |
| Milli-Q water | n/a | to 1 L |
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
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| DifcoTM tryptone peptone | 10 g/L | 10 g |
| BactoTM yeast extract | 5 g/L | 5 g |
| NaCl | 5 g/L | 5 g |
| Milli-Q water | n/a | to 1 L |
Dissolve 10 g of DifcoTM tryptone, 5 g of BactoTM 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. ChemiDocTM 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 SevenEasyTM (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
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文章信息
稿件历史记录
提交日期: Jul 21, 2026
接收日期: Oct 8, 2026
在线发布日期: Oct 30, 2026
出版日期: Nov 20, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Elmeeva, O. S., Smirnova, E. V., Rakitina, T. V., Kudriaeva, A. A. and Belogurov, A. A. (2026). Escherichia coli-Based Production and Purification From Inclusion Bodies of Hetero-Oligomeric Mitochondrial Protease ClpXP in Active Form. Bio-protocol 16(22): e5865. DOI: 10.21769/BioProtoc.5865.
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