Overview¶
Here, we make media used to grow up an expression strain to use for subsequent protein purification. We also make buffers used to purify those proteins using nickel histidine affinity chromatography.
Materials and Equipment¶
Bill of Materials
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| Potassium Hydroxide | Salts | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4°C to 30°C | link |
| Hydrochloric acid (1M) | Salts | Hydrochloric acid 1 N, Reagent Grade, 500 mL | VWR | E447-500L | $72.46 | 4°C to 30°C | link |
| HEPES | Salts | HEPES, Crystalline Powder, ≥99.5%, Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4°C to 30°C | link |
| Imidazole | Salts | ReagentPlus® Imidazole, Crystalline, 99% | Sigma-Aldrich | I202-500G | $120 | 4°C to 30°C | link |
| Ammonium Chloride | Salts | Ammonium chloride, ACS reagent, ≥99.5% | Sigma-Aldrich | 213330-500G | $73.50 | 4°C to 30°C | link |
| Sodium Chloride | Salts | Sodium Chloride, Redi-Dri™, anhydrous, ACS, ≥99% | Sigma-Aldrich | 746398-1KG | $133.00 | 4°C to 30°C | link |
| Potassium Chloride | Salts | Potassium Chloride, ACS reagent, 99.0-100.5% | Sigma-Aldrich | P3911-1KG | $146.00 | 4°C to 30°C | link |
| Magnesium Chloride hexahydrate | Salts | BioXtra MgCl₂·6H₂O, Powder or Crystals, ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | 4°C to 30°C | link |
| Glycerol | Supplements | Glycerol, Molecular Biology Grade, Liquid, ≥99.0% | Sigma-Aldrich | G5516-1L | $157.00 | 4°C to 30°C | link |
| Lysozyme | Supplements | Lysozyme from chicken egg white, ≥40,000 units/mg, lyophilized | Sigma-Aldrich | L6876-1G | $76.90 | -25°C to -15°C | link |
| TCEP-HCl (0.5M) | Supplements | Bond-Breaker® TCEP Neutral Solution, 5 mL | Thermo Scientific | 77720 | $175.65 | 4°C to 30°C | link |
| cOmplete Protease Inhibitor | Supplements | cOmplete™ Protease Inhibitor Cocktail, EDTA-Free, Tablets | Roche | 11873580001 | $472 | 1°C to 4°C | link |
| LB | Media | Luria Broth (Miller’s LB Broth), Molecular Biology Grade, Powder | Sigma-Aldrich | L3522-1KG | $221 | 4°C to 30°C | link |
| IPTG | Media | Isopropyl β-D-thiogalactoside, Powder, ≥99% (TLC) | Sigma-Aldrich | I6758-1G | $89.90 | -25°C to -15°C | link |
| Kanamycin | Media | Kanamycin sulfate, BioReagent, ≥750 µg/mg, Powder | Sigma-Aldrich | K1377-1G | $47.70 | 4°C to 30°C | link |
| 15 mL centrifuge tubes | Consumables | Corning® 15 mL PP Centrifuge Tubes, Sterile, 50/Rack | Corning | 430790 | $230.44 | 4°C to 30°C | link |
| 10 mL Luer lock syringes | Consumables | Syringes with BD Luer-Lok® Tip, 10 mL | BD Industrial/Difco | 550-80620-PK | $102.40 | 4°C to 30°C | link |
| 0.22 µm syringe filters | Consumables | PES Syringe Filter, 0.22µm, 30mm, Sterile | CELLTREAT | 229747 | $84 | 4°C to 30°C | link |
| 0.22 µm vacuum filters | Consumables | Corning® 500 mL Vacuum Filter, 0.22 µm PES Membrane, Sterile | Corning | 431097 | $187.14 | 4°C to 30°C | link |
Protocol¶
Step 1: Prepare Stock Buffers and Media¶
Make or buy the following stock solutions. Use ultrapure water (18.2 MΩ, e.g., Milli-Q).
| Stocks | Final Concentration (mM) | MW (g/mol) | Mass to add (g) | Final Vol (mL) | Storage (°C) |
|---|---|---|---|---|---|
| HEPES-KOH (pH 7.6) | 1000 | 238.3 | 238.3 | 1000 | room temp |
| Ammonium Chloride | 1000 | 53.49 | 53.49 | 1000 | room temp |
| Sodium Chloride | 5000 | 58.44 | 58.44 | 1000 | room temp |
| Potassium Chloride | 1000 | 74.55 | 74.55 | 1000 | room temp |
| Magnesium Chloride | 1000 | 203.3 | 203.3 | 1000 | room temp |
| Imidazole-HCl | 1000 | 68.08 | 68.08 | 1000 | room temp |
| Lysozyme | 30 mg/mL | - | 0.3 | 10 | -20°C |
| LB | 1x | - | 25 | 1000 | room temp |
| Kanamycin | 50 mg/mL (1000x) | - | 2.5 | 50 | -20°C |
| IPTG | 500 | 238.3 | 238.3 | 50 | -20°C |
Adjust pH of HEPES and Imidazole with Potassium Hydroxide and Hydrochloric Acid, respectively.
Note: prefer to use hydrated salts
Anhydrous Magnesium Chloride releases a lot of energy when dissolved in water, rapidly heating up the solution and causing violent bubbling and sputtering, which can burn you through gloves. Moreover, anhydrous salts can absorb water from the air, meaning that you have less salt than you’d expect in your final buffer.
Magnesium Chloride hydrates (as in, the Magnesium Chloride crystals incorporate water molecules into the crystal structure) do not release as much energy when dissolved and are much safer to work with. Moreover, they absorb less water over time, giving you more accurate buffer concentrations.
When calculating how much hydrated salt to use, don’t use the molecular weight of the anhydrous salt. You must consider the water weight as well. Hydrates generally have their adjusted molecular weights labelled on the bottle, but can be calculated by adding 18.02 g/mol for each water molecule.
Note: adjust pH then volume.
HEPES-KOH and Imidazole-HCl must be buffered (pH=7.6) using KOH and HCl, respectively.
First, resuspend HEPES or Imidazole in half the required volume of water. Next, using a pH meter and stir plate, measure pH while stirring. Slowly add KOH or HCl as appropriate to adjust pH to 7.6. Finally, transfer pH adjusted buffers to graduated cylinders and add water to target volume.
Note: store HEPES and Imidazole in the dark.
HEPES and Imidazole are light sensitive compounds! Keep stock solutions in the dark. Either wrap bottles in aluminum foil OR store bottles in a cabinet.
Step 2: Prepare Stable Buffers¶
Make the following buffers in advance and store at 4°C. Add all components except TCEP, which you MUST add fresh the day of use.
Wash Buffer — used to equilibrate columns and wash samples. Contains a small amount of Imidazole (20 mM) to reduce nonspecific protein binding to columns, improving purity.
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| HEPES-KOH (pH 7.6) | 50 | 1000 | 100 |
| Ammonium Chloride | 100 | 1000 | 200 |
| Sodium Chloride | 500 | 5000 | 200 |
| Magnesium Chloride | 10 | 1000 | 20 |
| Imidazole-HCl | 20 | 1000 | 40 |
| TCEP | 1 | 500 | 4 |
| Ultrapure water | — | — | 1436 |
| Total | 2000 |
Elution Buffer — used to elute proteins from Ni columns by introducing a large amount of Imidazole (500 mM) that competes with polyhistidine purification tags to bind the column.
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| HEPES-KOH (pH 7.6) | 50 | 1000 | 12.5 |
| Ammonium Chloride | 100 | 1000 | 25 |
| Sodium Chloride | 500 | 5000 | 25 |
| Magnesium Chloride | 10 | 1000 | 2.5 |
| Imidazole-HCl | 500 | 1000 | 125 |
| TCEP | 1 | 500 | 0.5 |
| Ultrapure water | — | — | 59.5 |
| Total | 250 |
Protein Buffer — used to remove Na+ and Imidazole from proteins. Filter with a 0.22 µm vacuum filter.
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| HEPES-KOH (pH 7.6) | 50 | 1000 | 25 |
| Potassium Chloride | 100 | 1000 | 50 |
| Magnesium Chloride | 10 | 1000 | 5 |
| TCEP | 1 | 500 | 1 |
| Ultrapure water | — | — | 419 |
| Total | 500 |
Protein Buffer (60% glycerol) — added to samples in Protein Buffer as a cryoprotectant for storage at -80°C. Filter with a 0.22 µm syringe filter. To use, add an equal volume of 60% glycerol buffer to samples in Protein Buffer (1:1).
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| HEPES-KOH (pH 7.6) | 50 | 1000 | 2.5 |
| Potassium Chloride | 100 | 1000 | 5 |
| Magnesium Chloride | 10 | 1000 | 0.5 |
| TCEP | 1 | 500 | 0.1 |
| glycerol | 60% | 100% | 30 |
| ultrapure water | — | — | 11.9 |
| Total | 50 |
Note: Protein Buffers are compatible with PURE reactions
The components of the PURE system are sensitive to the types and concentrations of the salts included in the reaction (e.g., [Mg2+] affects the relative rates of transcription vs translation). Proteins in Wash and Elution Buffer (high [NaCl] and [Imidazole]) cannot be used directly in PURE reactions because Na+ and Imidazole affect PURE function.
Protein Buffer (with and without glycerol) use K+ as a cation instead of Na+ and have no Imidazole.
Step 3: Prepare Working Buffers¶
Make the following buffers on the same day as use.
Lysis Buffer — used to resuspend bacterial pellets for lysis. Also enzymatically lyses cells with lysozyme (G. gallus). Contains protease inhibitor (cOmplete) to slow proteolysis.
| Reagent | Final Concentration (mM) | Stock Concentration (mM) | Volume to Add (mL) |
|---|---|---|---|
| HEPES-KOH (pH 7.6) | 50 | 1000 | 5 |
| Ammonium Chloride | 100 | 1000 | 10 |
| Sodium Chloride | 500 | 5000 | 10 |
| Magnesium Chloride | 10 | 1000 | 1 |
| Lysozyme | 0.3 mg/mL | 30 mg/mL | 1 |
| cOmplete Protease Inhibitor | 1 tablet / 50 mL | n/a | 2 tablets |
| TCEP | 1 | 500 | 0.2 |
| Ultrapure water | — | — | 72.8 |
| Total | 100 |
Note: Lysis Buffer is unstable at 4°C
We’ve observed Lysis Buffer stored at 4°C becoming cloudy over the span of one week. We believe this is due to lysozyme aggregating in solution. We suspect this affects the activity of the lysozyme, so we recommend preparing Lysis Buffer fresh for use the same day.
Downloads¶
References¶
Cell-free translation reconstituted with purified components
A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System
Acknowledgments¶
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), 751–755. 10.1038/90802
- Lavickova, B., & Maerkl, S. J. (2019). A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS Synthetic Biology, 8(2), 455–462. 10.1021/acssynbio.8b00427
- Grasemann, L., Lavickova, B., Elizondo-Cantú, M. C., & Maerkl, S. J. (2021). OnePot PURE Cell-Free System. Journal of Visualized Experiments, 172. 10.3791/62625