<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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You want to make PURE. You’re going to have to make Protein Mix, which contains the thirty-six (36) proteins in the PURE system. To make Protein Mix, you will need to:

1. Prepare buffers and gravity columns.
2. Grow cultures of E. coli expression strains for each protein.
3. Lyse each culture and purify target proteins by Ni2+ affinity column.
4. Measure the concentration and purity of each protein.
5. Combine proteins into a final Protein Mix.

This protocol shows you how to do each of these five (5) steps.

For more details, or if you want to look at specific subprotocols, see Subprotocols.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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• Table 1: Reagents and Consumables
• Table 2: Equipment

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 1: Prepare Materials

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• [ ] Prepare stock buffers and media.

  • [ ] Make or buy the following stock solutions. Use Ultrapure water (18.2 MOhm, e.g., Milli-Q).

• [ ] Prepare stable buffers and concentrates.

  Make the following buffers in advance and store at 4C. Add all components except TCEP, which you MUST add fresh the day of use.

  • [ ] P1 Wash Buffer - used to equilibrate columns and wash samples. P1 Wash Buffer contains a small amount of Imidazole (20 mM) to reduce nonspecific protein binding to columns, which improves the purity of your protein prep.

    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

  • [ ] P2 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

  • [ ] 4x P3 Concentrate - used to prepare P3 Exchange and P3 Storage Supplement later.

    Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
    HEPES-KOH (pH 7.6)	200	1000	100
    Potassium Chloride	400	1000	200
    Magnesium Chloride	40	1000	20
    Ultrapure water			180
    Total			500

    • Notes: P3 Buffers are compatible with PURE reactions

• [ ] Prepare Working Buffers (day of)

  • [ ] 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

    • Notes: Lysis Buffer is unstable at 4C

  • [ ] P3 Exchange Buffer - used to remove Na+ and Imidazole from proteins. Filter with a 0.22 um vacuum filter.

  Reagent	Final Concentration (mM)
  HEPES-KOH (pH 7.6)	50
  Potassium Chloride	100
  Magnesium Chloride	10
  TCEP	1

  Reagent	Volume to Add (mL)
  4x P3 Concentrate	125
  TCEP	1
  Ultrapure water	374
  Total	500

  • [ ] P3 Storage Supplement - added to samples in P3 Exchange Buffer as a cryoprotectant for storage at -80C. Filter P3 Storage Supplement with a 0.22 um syringe filter. To use, add an equal volume of P3 Storage Supplement to samples in P3 Exchange Buffer (1:1). P3 Exchange Buffer supplemented with P3 Storage Supplement is called “P3 Storage Buffer”.

• [ ] Prepare Columns.

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 2: Express Proteins in E. coli

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<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 3: Purify Proteins from E. coli

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• [ ] Lyse cells by sonication.
• [ ] Purify proteins by Ni2+ affinity column.
• [ ] Buffer exchange proteins into P3 Exchange Buffer.
• [ ] Store proteins until ready for use.