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 all the materials that you will need,
express each protein in an E. coli expression strain,
purify each protein (by Ni-His affinity columns),
quantify their concentration and purity, and
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

Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Columns
Gravity columns	Empty Disposable PD-10 Columns	Cytiva	17043501	$258.30	4C to 30C	[link]
Ni-His Resin	NEBExpress Ni Resin	New England Biolabs	S1428S	$358.00	1C to 4C	[link]

Salts (for Buffers)
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
Hydrochloric acid (1M)	Hydrochloric acid 1 N, Reagent Grade, Packaging=Amber Bottle, Size=500 mL	VWR	E447-500L	$72.46	4C to 30C	[link]
HEPES	HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle	Sigma-Aldrich	H3375-500G	$431	4C to 30C	[link]
Imidazole	ReagentPlus® Imidazole, Crystalline, 99%	Sigma-Aldrich	I202-500G	$120	4C to 30C	[link]
Ammonium Chloride	Ammonium chloride,ACS reagent, ≥99.5%	Sigma-Aldrich	213330-500G	$73.50	4C to 30C	[link]
Sodium Chloride	Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99%	Sigma-Aldrich	746398-1KG	$133.00	4C to 30C	[link]
Potassium Chloride	Potassium Chloride, ACS reagent, 99.0-100.5%	Sigma-Aldrich	P3911-1KG	$146.00	4C to 30C	[link]
Magnesium Chloride hexahydrate	BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0%	Sigma-Aldrich	M2670-100G	$50.50	4C to 30C	[link]

Supplements (for Buffers)
Glycerol	Glycerol, Molecular Biology Grade, Liquid, ≥99.0%	Sigma-Aldrich	G5516-1L	$157.00	4C to 30C	[link]
Lysozyme	Lysozyme from chicken egg white, protein ≥90%, ≥40,000 units/mg protein, lyophilized powder	Sigma-Aldrich	L6876-1G	$76.90	-25C to -15C	[link]
TCEP-HCl (0.5M; presuspended)	Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL	Thermo Scientific	77720	$175.65	4C to 30C	[link]
cOmplete Protease Inhibitor	cOmplete™ Protease Inhibitor Cocktail, EDTA-Free, Tablets	Roche	11873580001	$472	1C to 4C	[link]

Media
LB	Luria Broth (Miller's LB Broth), Non-Sterile, 6.8 - 7.2, Molecular Biology Grade, suitable for regular E.coli culture, Powder	Sigma-Aldrich	L3522-1KG	$221	4C to 30C	[link]
IPTG	Isopropyl β-D-thiogalactoside (IPTG), Powder, ≥99% (TLC), ≤0.1% Dioxane	Sigma-Aldrich	I6758-1G	$89.90	-25C to -15C	[link]
Kanamycin	BioReagent Kanamycin sulfate, ≥750 ug/mg, From Streptomyces kanamyceticus, Suitable for cell culture, Suitable for plant cell culture, Powder	Sigma-Aldrich	K1377-1G	$47.70	4C to 30C	[link]
Misc. Reagents
Pierce660 Reagent	Pierce™ 660nm Protein Assay Reagent	Thermo Scientific	22660	$176.65	4C to 30C	[link]
BSA Protein Standard	Pierce™ Bovine Serum Albumin Standard Ampules, 2 mg/mL	Thermo Scientific	23209	$81.65	4C to 30C	[link]

Consumables
Culture tubes	Culture Tube, PS, 14mL, 18x95mm, Sterile, TC Treated, w/ Snap (Vent) Cap	Greiner Bio-One	191160	$258.15	4C to 30C	[link]
15 mL centrifuge tubes	Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case	Corning	430790	$230.44	4C to 30C	[link]
10 mL Luer lock syringes	Syringes with BD Luer-Lok® Tip, BD Medical, Syringe with Luer-Lok® Tip, Volume=10 mL	BD Industrial/Difco	550-80620-PK	$102.40	4C to 30C	[link]
0.22 um syringe filters	PES Syringe Filter, 0.22μm, 30mm, Sterile	CELLTREAT Scientific Products	229747	$84	4C to 30C	[link]
0.22 um vacuum filters	Corning® 500 mL Vacuum Filter/Storage Bottle System, 0.22 µm Pore 33.2cm² PES Membrane, Sterile, 12/Case	Corning	431097	$187.14	4C to 30C	[link]
0.45 um syringe filters	PES Syringe Filter, 0.45μm, 30mm, Sterile	CELLTREAT Scientific Products	229749	$84	4C to 30C	[link]
15 mL spin filters (3 kDa cutoff)	Amicon® Ultra Centrifugal Filter, 3 kDa MWCO	Millipore Sigma	UFC9003	$125	4C to 30C	[link]
0.5 mL spin filters (3 kDa cutoff)	Amicon® Ultra Centrifugal Filter, 3 kDa MWCO	Millipore Sigma	UFC5003	$63	4C to 30C	[link]
Tris-Glycine 10—20% gels	Novex™ Tris-Glycine Mini Protein Gels, 10–20%, 1.0 mm, WedgeWell™ format	Invitrogen	XP10205BOX	$120.65	1C to 4C	[link]
96-well optical plate	Microplate, 96 well, PS, U-bottom, clear	Greiner	650101	$156.14	4C to 30C	[link]

Glassware
250 mL baffled flasks	PYREX® 250 mL Delong Shaker Erlenmeyer Flask with Baffles	Pyrex	4444-250	$188.26	4C to 30C	[link]
flask closures	Chemglass Life Sciences Closure, 38mm, Stainless Steel	Chemglass Life Sciences	Chemglass Life Sciences Closure, 38mm, Stainless Steel	$100.75	4C to 30C	[link]

Equipment
Sonicator	Q700 Sonicator	QSonica	Q700A-110	$5500	4C to 30C	[link]
Sonicator Probe	Standard Probe, Replaceable Tip, 1/2” (12.7mm) diameter	QSonica	4220	$670	4C to 30C	[link]
Sound Enclosure	Sound Enclosure	QSonica	432B2	$995	4C to 30C	[link]
Lab Jack	Fisherbrand™ Lab Jacks (15cmx15cm)	Fisher Scientific	14-673-51	$484	4C to 30C	[link]
Gel Tank	Mini Gel Tank	Thermo Fisher	A25977	$664.65	4C to 30C	[link]
Gel Power Supply	PowerEase™ Touch Power Supply	Invitrogen	PS0120	$861.65	4C to 30C	[link]
Plate Reader	BioTek Cytation 5 Cell Imaging Multimode Reader	Agilent	CYT5MFAWSN	Must request quote	4C to 30C	[link]

Step 1: Prepare Materials for Purification

Make (or buy) the following stock solutions. Use deionized water (e.g., milliQ).

Reagent	MW [g/mol]	Amount [g]	Final Volume [mL]	Storage	Needs pH adjustment?	Needs Sterilization?
Stock Solutions
Potassium Hydroxide (1M)	56.11	14.0	250	4C to 30C	no	no
HEPES-KOH (pH=7.6; 1M)	238.3	59.5	250	4C to 30C; dark	yes	no
Imidazole-HCl (pH=7.6; 1M)	68.08	34.0	500	4C to 30C; dark	yes	no
Ammonium Chloride (1M)	53.49	13.4	250	4C to 30C	no	no
Sodium Chloride (5M)	58.44	73.1	250	4C to 30C	no	no
Potassium Chloride (1M)	74.55	18.6	250	4C to 30C	no	no
Magnesium Chloride (1M)	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 (0.5 M)	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)

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Notes: use hydrated salts when able to and avoid explosions!

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Notes: adjust pH then volume.

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Notes: store HEPES and Imidazole in the dark.

Make the following buffers in advance and store at 4C:

P1 Wash Buffer - used to equilibrate all columns and wash samples. P1 Wash Buffer contains a small amount of imidazole (20 mM) to reduce nonspecific protein binding to columns.

Reagent	Final Concentration [mM]	Stock Concentration [mM]	Volume to Add [mL]
HEPES-KOH	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
water (milliQ)			1436
Total			2000

P2 Elution Buffer - used to elute proteins from Ni-His columns. P2 Elution Buffer contains a large amount of imidazole (500 mM) to displace 6xHis tagged proteins from Ni-His columns.

Reagent	Final Concentration [mM]	Stock Concentration [mM]	Volume to Add [mL]
HEPES-KOH	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
water (milliQ)			59.5
TCEP	1	500	0.5
Total			250

4x P3 Concentrate - used to prepare P3 Exchange and P3 Storage buffers later.

Reagent	Volume to Add [mL]	Final Concentration [mM]	Stock Concentration [mM]
4x P3 Concentrate
HEPES-KOH	100	200	1000
Potassium Chloride	200	400	1000
Magnesium Chloride	20	40	1000
water (milliQ)	180
Total	500

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Notes: P3 Buffers are compatible with PURE reactions

Make the following buffers on the same day as use:

Lysis Buffer - used to resuspend bacterial pellets for mechanical lysis by sonication. Also chemically 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	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
TCEP	1	500	0.2
cOmplete Protease Inhibitor	1 tablet / 50 mL	n/a	2 tablets
water (milliQ)			72.8
Total			100

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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	50
Potassium Chloride	100
Magnesium Chloride	10
TCEP	1

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

P3 Storage Supplement - used to protect samples in P3 Exchange buffer from damage by freezing during storage at -80C. Add an equal volume of P3 Storage Supplement to samples in P3 Exchange Buffer (1:1). Filter P3 Storage Supplement with a 0.22 um syringe filter.

Reagent	Final Concentration [mM]
HEPES-KOH	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
water (milliQ)	1.48
Total	10

Store all buffer concentrates at 4C until ready to use (up to one month).

Prepare Columns.

Pack columns.

We used PD-10 gravity columns, which came with ~ 12 mL gravity columns, plastic filters (to hold the resin bed), plastic bungs (caps for column tips) and plastic lids (caps for column openings).

Cut column tips off using scissors or a razor blade.

Pack a filter into the bottom of the column using the back end of a cell spreader.

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Notes: the columns have ridges!

Wash empty columns.

Attach columns to a lab stand by clamp above a beaker (≥ 500 mL) to capture waste flowthrough.

Wash columns and filters with 5 CV of milliQ water; allow water to flow through.

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Notes: “CV” = Column Volume

Load and Equilibrate Ni-His Resin into Ni-His columns.

Resuspend Ni-His resin (50% v/v suspension) by shaking bottle.

Add 2 mL resin by pipette to each column.

Equilibrate columns with 10 CV (20 mL) of cold P1 Wash Buffer (4C); allow buffer to flow through.

Store Columns

Seal each column with a cap and bung.

Store columns at 4C until ready for use.

Step 2: Express Proteins in E. coli

Prep overnight cultures.

Add 5 mL LB + kanamycin (50 ug / mL) to 15 mL culture tubes. Label each tube.

Poke your expression strain working stock with a pipette tip.

Eject the whole tip into each culture tube.

Incubate cultures overnight at 37C / 250 rpm for between 10 hrs and 16 hrs.

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Notes - you can work from glycerol stocks OR fresh colonies.

Perform bulk outgrowth.

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

Incubate back diluted cultures at 37C / 250 rpm / to mid-log phase (OD600 between 0.4 and 0.6 ~ 3.5 hrs.)

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Notes: leave ≥ 2.5x culture volume in headroom!

Induce protein expression.

Once bacterial cultures reach midlog phase (OD600 between 0.4 and 0.6) add IPTG to 500 uM. To do this, we added IPTG from 0.5M aliquots (1000x) at 1:1000. E.g., for 100 mL of culture, we add 100 uL of IPTG at 0.5M.

Incubate induced cultures at 37C / 250 rpm / 4 hr to allow cells to express proteins.

Centrifuge cells and freeze pellets.

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

Pour 50 mL of each culture into 50 mL centrifuge tubes and label each (2x50 mL tubes per 100 mL culture).

Centrifuge cultures at 3200 rcf / 4C / 30 min.

Decant supernatant and reserve pellets.

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

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Notes: freezing pellets may help lyse cells.

Step 3: Purify Proteins from E. coli

Lyse cells by sonication.

Prepare centrifuge and lysis buffer.

Prechill centrifuge and lysis buffer to 4C.

Finish lysis buffer with TCEP (500x, or 1 mL TCEP per 2 L buffer).

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Notes - keep lysate cold to prevent proteolysis!

Resuspend pellets in cold lysis buffer.

Thaw bacterial pellets on ice.

Add 10 mL lysis buffer to each pellet. Resuspend pellets completely by vortexing.

Transfer resuspended pellets to 15 mL centrifuge tubes

Lyse cells by sonication.

Set sonicator to appropriate setting. We lysed cells to a final energy of 12 000J / 40 mL total lysate (50% amplitude, 20s on / 20s s off).

Put resuspended pellets in an ice bucket. Place samples on a lab jack beneath the sonicator tip(s).

Adjust sample height using the lab jack such that the sonicator tip(s) are submerged about one quarter (1/4) of the way into the sample (e.g., 2.5 mL into a 10 mL sample).

Lyse cells by sonication.

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Notes - always use a Sound Enclosure during sonication.

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Notes - avoid foaming during sonication.

Clarify lysates by centrifugation and filtration.

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

(Meanwhile) for each lysate, assemble 10 mL Luer lock syringes and filters (we used 0.45 um for lysates). Pull the plunger out of the syringe, screw on the filter, and leave the plunger outside of the syringe.

For each lysate, decant the supernatant into syringe and put in the plunger. Plunge lysate through filter into a collection tube (we used 15 mL centrifuge tubes).

Purify proteins by Ni-His affinity column.

Wash each column with 5 column volumes (”CVs”) of DI water (i.e., 5x 2mL resin bed = 10 mL DI water).

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

Load up to 5 CVs (10 mL) clarified lysate per column and allow to flow through. (Optionally) capture flowthrough for later analysis; see

Pierce660 Assay and

Protein Gel.

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Notes: you can load more than 10 mL lysate in tranches.

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

Pierce660 Assay and

Protein Gel.

Elute sample in 2.5 mL cold P2 buffer. Capture flow through in 15 mL centrifuge tube.

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Notes: the first 500 uL fraction has little protein.

Exchange proteins from P2 Elution Buffer to P3 Exchange Buffer.

Dilute sample ~ 10x with the final buffer(e.g., 5 mL protein in P2 elution buffer + 45 mL P3 Exchange Buffer).

Load sample onto the spin column.

Centrifuge samples at 4000 rcf for 10 min at 4C.

Repeat loading and spinning until all the sample has been processed.

Dilute sample ≥ 10x further with the final buffer and repeat loading and spinning until all the sample has been processed.

Store proteins until ready for use.

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: Quantify Protein Yield and Purity

Assess 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.

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.

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Notes: we do 2x serial dilutions

Mix Pierce660 Reagent well before use by inversion.

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 in column 1, …) to the optical plate.

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

Incubate plate at 25C for 5 minutes. Samples should turn green.

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

Analyze results.

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

Prepare the standard curve by plotting the background subtracted absorbance vs. concentration for each BSA sample.

Fit the standard curve by linear regression.

For each dilution series of each sample, choose a dilution in the range of the standard curve.

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

Assess protein purity by protein gel.

Denature samples.

Label one tube per sample.

Add 7 uL of 4x sample buffer to each tube.

Add 21 uL of each sample to its tube. Mix by pipetting.

Incubate samples at 90C / 10 min.

Set up gel box.

Open an individually wrapped protein gel (we use 10% - 20% Tris Gly gels). Throw out the bag that the gel came with.

Remove plastic tape at bottom of gel. This exposes a strip of gel to buffer, allowing current to flow through the gel and out the bottom.

Place gel in gel box and seal tightly.

Pour running buffer (we use Tris Gly) into the front half of the gel box reservoir. Check to make sure that the gel is tightly sealed (i.e., no buffer can run from the front to the back of the gel box).

Pour running buffer into the back half of the gel box reservoir.

Remove comb from top of gel.

Load between 10 uL and 20 uL of sample onto each lane of the gel. Remember to include a protein ladder!

Run the gel. We use 150 V / 60 min.

Stain and destain the gel.

Using a gel knife (looks like a paint can opener), pop open the plastic cassette holding the gel.

Poke the gel out of the plastic box by pushing the gel knife through the exposed strip of gel on the back of the plastic cassette. This step is easier if the gel knife is wet.

Briefly rinse the gel in milliQ water.

Submerge gel in a rapid staining protein stain in a waterproof container. Incubate gel at room temperature with rocking for up to 1 hr.

Decant staining solution, rinse gel at least once, then submerge gel in milliQ water. Incubate gel at room temperature with rocking for 15 min.

Repeat above step until bands are clearly visible (we do three destaining steps).

Observe gel.

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Note: use an imager to semi-quantitate!

Step 5: Assemble Protein Mix

Calculate how much volume of each protein stock is needed for a given final volume of Protein Mix (here: 100 uL)

#	Protein	Protein Mix Concentration [ng/uL]	Stock Concentration [ng/uL]	Volume to Add per 100 uL Protein Mix [uL]
1	AlaRS	538
2	ArgRS	15
3	AsnRS	169
4	AspRS	62
5	CysRS	9
6	GlnRS	29
7	GluRS	97
8	GlyRS	74
9	HisRS	6
10	IleRS	308
11	LeuRS	31
12	LysRS	49
13	MetRS	18
14	PheRS	131
15	ProRS	77
16	SerRS	15
17	ThrRS	48
18	TrpRS	48
19	TyrRS	5
20	ValRS	14
21	IF1	77
22	IF2	308
23	IF3	77
24	EF-G	385
25	EF-Tu	3846
26	EF-Ts	385
27	RF1	77
28	RF2	77
29	RF3	77
30	RRF	77
31	MTF	154
32	CK	31
33	MK	23
34	NDK	8
35	PPiase	8
36	T7RNAP	77
	Total	7430

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

Aliquot Protein Mix (e.g., eight (8) PCR tubes of 12.5 uL each) and store at -80C until ready for use.

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 cell lysate for purification from frozen cell pellets:

Lyse Bacteria by Sonication

Purify proteins from cell lysate:

Purify Proteins by Ni-His 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