Make Protein Mix

Getting Started

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:

Prepare buffers and gravity columns.
Grow cultures of E. coli expression strains for each protein.
Lyse each culture and purify target proteins by Ni2+ affinity column.
Measure the concentration and purity of each protein.
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 Make Protein Mix - Subprotocols.

Materials and Equipment

‣

Table 1: Reagents and Consumables

‣

Table 2: Equipment

Step 1: Prepare Materials

Prepare stock buffers and media.

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

Reagent	MW (g/mol)	Mass (g)	Final Volume (mL)	Storage	Adjust pH?	Sterilize?
Stock Solutions
Potassium Hydroxide (1 M)	56.11	14.0	250	4C to 30C	no	no
HEPES-KOH (1 M, pH 7.6)	238.3	59.5	250	4C to 30C; dark	yes	no
Imidazole-HCl (1 M, pH 7.6)	68.08	34.0	500	4C to 30C; dark	yes	no
Ammonium Chloride (1 M)	53.49	13.4	250	4C to 30C	no	no
Sodium Chloride (5 M)	58.44	73.1	250	4C to 30C	no	no
Potassium Chloride (1 M)	74.55	18.6	250	4C to 30C	no	no
Magnesium Chloride (1 M)	203.3	20.3	100	4C to 30C	no	no
Lysozyme (30 mg/mL)	14320	1	33	-25C to -15C	no	no

Media
LB	n/a	25	1000	4C to 30C		autoclave OR filter (0.22 um)
IPTG (500 mM)	238.3	1	4.2	-25C to -15C	no	filter (0.22 um)
Kanamycin (50 mg/mL)	484.5	2.5	50	-25C to -15C	no	filter (0.22 um)

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”.

Reagent	Final Concentration (mM)
HEPES-KOH (pH 7.6)	50
Potassium Chloride	100
Magnesium Chloride	10
TCEP	1
Glycerol	60% (v/v)

Reagent	Volume to Add (mL)
4x P3 Concentrate	2.5
Glycerol	6.0
TCEP (0.5 M)	0.020
Ultrapure water	1.48
Total	10

Prepare Columns.

Assemble columns.

Cut tips off of columns off using scissors or a razor blade.

Pack a filter into the bottom of the column (we use the back end of a cell spreader).

‣

Notes: columns have ridges!

Wash empty columns.

Clamp columns to a lab stand and put a large beaker (≥ 500 mL) underneath to capture flowthrough.

Wash columns and filters with 10 mL Ultrapure water.

Load and Equilibrate Ni2+ affinity Resin into columns

Resuspend Ni2+ resin (50% v/v suspension) by shaking.

Add 2 mL resin by pipette to each column.

Equilibrate columns with 10 CV of cold P1 Wash Buffer (4C) and discard flowthrough.

‣

Notes: “CV” = Column Volume

Store Columns.

Seal each column with a cap and bung.

Store columns at 4C for up to 48 hrs.

‣

Notes: store columns long term in EtOH (20%)

Step 2: Express Proteins in E. coli

Prep overnight cultures.

Add 5 mL LB + Kanamycin (50 ug / mL) to 15 mL culture tubes and label.

Innoculate your tubes with your expression strain working stock using a pipette tip.

‣

Notes - you can use glycerol stocks OR fresh colonies as working stocks

Incubate cultures overnight at 37C / 225 rpm for between 12 hrs and 16 hrs.

Perform bulk outgrowth.

Back dilute overnight cultures 1:1000 into fresh media (e.g., add 100 uL of overnight and 100 mL LB with Kanamycin to 250 mL Erlenmeyer flasks).

‣

Notes: leave ≥ 2.5x culture volume in headroom!

Incubate back diluted cultures at 37C / 225 rpm / to mid-log phase (OD600 between 0.4 and 0.6 ~ 3.5 hrs).

Induce protein expression.

At mid-log phase, induce your cultures with IPTG to 500 uM (e.g., add 100 uL of IPTG (0.5M) to a 100 mL culture).

Incubate induced cultures at 37C / 225 rpm / 4 hr to allow cells to express proteins.

Centrifuge cells and freeze pellets.

While incubating your induced cultures, pre-chill your centrifuge and rotor to 4C.

Harvest your cultures by centrifuging at 3200 rcf / 4C / 30 min.

Decant supernatant and reserve pellets.

Weigh pellets to calculate your biomass yield (gDCM / L).

Store pellets at -80C and allow to freeze (at least overnight)

‣

Notes: take a break!

Step 3: Purify Proteins from E. coli

Lyse cells by sonication.

Prepare centrifuge and Lysis Buffer.

Pre-chill your centrifuge and Lysis Buffer to 4C.

Finish Lysis Buffer with TCEP (0.5M) (e.g., add 500 uL TCEP to 1 L Lysis Buffer).

Resuspend pellets in cold Lysis Buffer.

Thaw bacterial pellets on ice.

Weigh 1 g dry cell mass per pellet.

Resuspend 1 g pellets in 10 mL Lysis Buffer by vortexing.

‣

Notes - keep lysate cold to prevent proteolysis.

Lyse cells by sonication.

Program your sonicator. We recommend 50% amplitude, 10s on / 10s off.

‣

Warning - always use a Sound Enclosure during sonication!

Place resuspended samples under the sonicator horn in an ice bucket on a lab jack.

Adjust your sample height using the sample jack until each sonicator tip is submerged by about one quarter (1/4) of the sample height (e.g., 2.5 mL into a 10 mL sample).

Lyse cells by sonication. Deliver 4.5 kJ for every 10 mL sample (e.g., a four-pronged sonicator horn lysing 4x 10 mL samples → 18 kJ).

‣

Notes - avoid foaming during sonication.

Clarify lysates by centrifugation and filtration.

Centrifuge lysates at 4 krcf / 4C / 45 min.

(Meanwhile) for each lysate, assemble a 10 mL Luer lock syringe and 0.22 um syringe filter.

Carefully decant each supernatant into a syringe and filter into a fresh 15 mL centrifuge tube. Keep clarified lysate cold (4C).

‣

Notes - keep going! Don’t take a break!

Purify proteins by Ni2+ affinity column.

Wash each column with 5 CV of Ultrapure water. For this protocol, we will assume you are using a 2 mL resin bed.

‣

Notes: “CV” = Column Volume

Equilibrate each column with ≥ 5 CV (10 mL) cold P1 buffer.

Load up to 5 CV (10 mL) clarified lysate per column. (Optionally) capture flowthrough for later analysis (see

Pierce660 Assay and

Protein Gel).

‣

Notes: if you have >10 mL lysate, load in tranches.

Wash column with ≥ 5 CV (10 mL) cold P1 buffer. Allow buffer to flow through. (Optionally) capture flowthrough for later analysis; see

Pierce660 Assay and

Protein Gel.

Elute sample with 3 CV (6 mL) cold P2 buffer. Capture flow through in 15 mL centrifuge tube.

Buffer exchange proteins into P3 Exchange Buffer.

Here, use P3 Exchange Buffer as your new buffer.

Dilute your sample ~10x with your new buffer (e.g., add 54 mL P3 Exchange Buffer to 6 mL eluted protein).

Load your sample onto a centrifugal filter column.

‣

Notes: use filter cutoffs 3x smaller than your sample.

Centrifuge samples at either 4000 rcf (4 mL or 15 mL filters) or 14 000 rcf (0.5 mL filters) at 4C until you’ve reached your target volume. Check on your sample volume in the first 10 min, then again as needed.

‣

Notes: if you have too much sample, load in tranches.

Continue to dilute and spin your sample, noting your sample volume and dilution volume at each step, until you’ve reached your target dilution factor (we recommend ≥ 300x).

‣

Notes: how to calculate dilution factor.

Store columns for later use.

Wash columns by loading with ddH2O and spinning.

Load columns with EtOH 20% (v/v) and store at room temp.

‣

Notes: If you see precipitate, dilute your sample immediately.

Store proteins until ready for use.

Visually estimate the total volume of protein, or measure by reverse-pipette.

‣

Notes: how to reverse pipette.

Add an equal volume of P3 Storage Supplement to each protein sample (e.g., 100 uL P3 Storage Supplement to 100 uL EF-Tu in P3 Exchange Buffer).

Mix gently by pipetting.

Store frozen at -80C until ready for use.

Step 4: Measure Protein Yield and Purity

Measure your protein concentration by Pierce660 Assay.

Prepare a standard curve within the assay’s working range (125 ug / mL to 2000 ug / mL). Remember to dilute the BSA stock in the same buffer used for your sample. The standards can be stored at -20C for future assays.

Concentration	Volume of BSA Stock (2 mg/mL)	Volume of Buffer
2 mg/mL	200 uL	0 uL
1.5 mg/mL	150 uL	50 uL
1 mg/mL	100 uL	100 uL
0.75 mg/mL	75 uL	125 uL
0.50 mg/mL	50 uL	150 uL
0.25 mg/mL	25 uL	175 uL
0.125 mg/mL	12.5 uL	187.5 uL
0 mg/mL	0	200 uL

Prepare a dilution series of your samples in the same buffer.

‣

Notes: we do 2x serial dilutions

Mix Pierce660 Reagent well by inverting the bottle before use.

Array 150 uL of Pierce660 Reagent on a 96-well optical plate.

Add 10 uL of each sample (BSA standard series and sample concentration series) column-wise (e.g., BSA standard in Column 12, Sample 1 series in column 1, …) to the optical plate.

‣

Notes: adjust your sample volume as needed

Cover your plate with aluminum foil and mix on a plate shaker at medium speed for 1 minute.

Incubate your plate at 25C / 5 min. Samples should turn from brown to green.

Using a plate reader, measure the absorbance of the samples at 660 nm.

Analyze results.

Subtract the absorbance of blank samples (i.e., BSA standard = 0 mg / uL) from all other samples (”background subtracted absorbance”).

Plot the standard curve by plotting the background subtracted absorbance vs. concentration for each BSA standard. Fit a line to your standard curve.

For each sample dilution series, choose a well with a background subtracted absorbance in the linear range of the standard curve.

Using the linear fit of your standard curve, calculate the concentration of the sample.

Measure protein purity by protein gel.

Denature samples.

Label one tube per sample.

Add 4 uL of 4x sample buffer and 16 uL of sample (~0.1-0.2 ug / uL) per tube. Mix by pipetting.

Incubate samples at 90C / 10 min.

Set up a gel box.

Open an individually wrapped protein gel (we use 10% - 20% Tris Gly gels).

Remove plastic tape at the bottom of gel. This exposes a strip of the gel to the running buffer, allowing current to flow from one electrode through the gel to the other electrode.

Place gel in gel box and seal tightly. Check the seal by pouring running buffer (we use Tris Gly) into the front half of the reservoir and checking that no buffer leaks into the back half. If so, remove the gel, empty the reservoir and try again.

Pour running buffer into the back half of the reservoir.

Carefully, remove comb from top of gel.

Load 10 uL protein ladder onto the outermost lanes.

Load each sample onto its own lane. Target between 1-2 ug purified protein and between 10 uL and 20 uL per lane.

Run the gel at 200 V / 40-50 min, or until the dye front touches the bottom of the gel.

Stain and destain the gel.

Using a gel knife, crack open the plastic cassette holding the gel.

Remove the gel carefully into a waterproof container. We find this is easier if you cover the bottom of your container with Ultrapure water (≥ 3 mm).

Cover the gel with Ultrapure water (~40 mL), microwave for 30 seconds, and rock for 5 minutes. Decant and repeat this step twice, washing a total of three (3) times.

Cover gel in a rapid protein stain (~40 mL) in the same container. We use PageBlue Protein Staining Solution. Microwave for 30 seconds, then incubate at room temperature with rocking for ~30 minutes.

Decant staining solution, rinse gel at least once, then cover in Ultrapure water. Incubate gel at room temperature with rocking until you can see distinct protein bands (up to overnight).

‣

Notes: Use Kimwipes to speed up destaining

Observe gel. High purity protein preps should have distinct bands at the correct molecular weight, with no unexpected additional bands.

‣

Notes: you can use a gel imager to semi-quantitate your protein

Step 5: Assemble Protein Mix

Read the following Google Sheet template and follow its directions to assemble your Protein Mix. https://docs.google.com/spreadsheets/d/1OLFNVuiL5-f6FAD-eY3rEHRrYya3oPlh2JK1UhwY7qs/edit?usp=sharing

Untitled

Input the following information into blue colored cells:

Desired number of 10 uL PURE reactions

Protein stock concentrations

Protein stock volumes

Check the “Checklist” section to see if any cells are highlighted yellow. If so, follow the directions next to the highlighted cell.

‣

Notes: if your Protein Mix is dilute, you can concentrate it

Concentrate Protein Mix to the Target Final Volume (~ 12.25 mg / mL) using a 3kDa Amicon Centrifugal Filter.

‣

Notes: wash and concentrate your Protein Mix after assembly

Measure the total protein concentration of your Protein Mix:

Create 10x dilutions of Protein Mix (1 uL in 9 uL Ultrapure water) in triplicate.

Perform a

Pierce660 Assay on your diluted Protein Mix samples.

Calculate the total protein concentration of your diluted Protein Mix samples. Average the values of each triplicate and multiply by 10 to estimate the total protein concentration of your Protein Mix.

Dilute your Protein Mix to 12.25 mg / mL in P3 Storage Buffer (30% glycerol). If the concentration of your Protein Mix is less than 12.25 mg / mL, repeat the last two steps (concentrate your mix, then measure using Pierce660)

Aliquot your Protein Mix (12 uL + 1 uL of head room per tube) into PCR tubes or microcentrifuge tubes and store -80C.

Subprotocols

Prepare buffers, media, and columns for purification:

Prepare Columns and

Make Protein Purification Buffers and Media

Express proteins for purification:

Grow and Induce Expression Strains

Prepare clarified lysate from cell pellets for purification:

Lyse Bacteria by Sonication

Purify proteins from clarified lysate:

Purify Proteins by Ni2+ Gravity Column

Get your purified proteins in the right buffer at the right concentration:

Exchange Buffers and Concentrate by Spin Filtration

Measure the yield and purity of your proteins:

Protein Gel and

Pierce660 Assay

Assemble Protein Mix from purified proteins:

Assemble Protein Mix

Resources and References

Papers

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

Cell-free translation reconstituted with purified components

OnePot PURE

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription–translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

doi.org

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

OnePot PURE Cell-Free System | Text Page

Scientific Article | The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for...

dx.doi.org

OnePot PURE Cell-Free System | Text Page

Credits

Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)

Developers