Make Ribosomes

Getting Started

Ribosomes are large complexes of RNA and proteins (MW ~2.7 MDa) that are the sites of protein synthesis. Ribosomes coordinate the decoding of mRNA transcripts by tRNA and catalyze the formation of each peptide bond in new proteins, making ribosomes a key component of any protein synthesis system. Ribosomes can be purified from E. coli biomass by a variety of methods (e.g., His-tagged ribosomes can be purified by Ni-His chromatography, as in Purify Proteins by Ni2+ Gravity Column), but we recommend a two step process: (1) initial, tag-free purification by hydrophobic interaction chromatography (HIC) and (2) size-selective precipitation by ultracentrifugation. This protocol will show you how to grow E. coli A19 biomass and purify ribosomes by HIC followed by ultracentrifugation.

Materials and Equipment

‼️All reagents and materials must be prepared RNase-free. Use RNaseZap or 10% bleach to decontaminate plastic and glassware and rinse with nuclease-free water. We find ultrapure water (18.2 MOhm) is often sufficient for RNase-free work.

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Table 1: Reagents and Consumables

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Table 2: Equipment

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Table 3: Stock buffers

Protocol

Prepare Buffers and Media

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

Buffer Template: Unsupported Embed

Ribosome Lysis Buffer

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
HEPES-KOH (1 M, pH 7.6)	10	1000	2.5
Magnesium Acetate (1 M)	10	1000	2.5
Potassium Chloride (1 M)	50	1000	2.5
TCEP-HCl (0.5M)	1	500	0.5
Ultrapure water	-	-	242
Total			250

Salting Out Buffer

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
HEPES-KOH (1 M, pH 7.6)	10	1000	1
Magnesium Acetate (1 M)	10	1000	1
Potassium Chloride (1 M)	50	1000	1
Ammonium Sulfate	3000	n/a	39.6 g
TCEP-HCl (0.5M)	1	500	0.2
Ultrapure water	-	-	92.8
Total			100

Ribosome Wash Buffer

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
HEPES-KOH (1 M, pH 7.6)	20	1000	10
Magnesium Acetate (1 M)	10	1000	5
Ammonium Sulfate	1500	n/a	99.1 g
TCEP-HCl (0.5M)	1	500	1
Ultrapure water	-	-	484
Total			500

Ribosome Elution Buffer

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
HEPES-KOH (1 M, pH 7.6)	20	1000	6
Magnesium Acetate (1 M)	10	1000	3
TCEP-HCl (0.5M)	1	500	0.6
Ultrapure water	-	-	290.4
Total			300

Cushion Buffer

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
HEPES-KOH (1 M, pH 7.6)	20	1000	5
Magnesium Acetate (1 M)	10	1000	2.5
Ammonium Chloride (1 M)	30	1000	7.5
Sucrose	30% (w/v)	n/a	75 g
TCEP-HCl (0.5M)	1	500	0.5
Ultrapure water	-	-	234.5
Total			250

Ribosome Buffer

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
HEPES-KOH (1 M, pH 7.6)	20	1000	5
Magnesium Acetate (1 M)	6	1000	1.5
Potassium Chloride (1 M)	30	1000	7.5
TCEP-HCl (0.5M)	1	500	0.5
Ultrapure water	-	-	235.5
Total			250

AcOH (0.1 M)

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
Acetic Acid (glacial; 17.4M)	100	17400	1.44
Ultrapure water	-	-	248.5
Total			250

EtOH (20% v/v)

Reagent	Final Concentration (mM)	Stock Concentration (mM)	Volume to Add (mL)
Ethanol	20% v/v	100% v/v	50
Ultrapure water	-	-	200
Total			250

Cell culture:

Prepare overnight cultures.

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Notes - We purify Ribosomes from A19

Add 5 mL Luria Broth (LB) under sterile conditions to two (2) 14 mL culture tubes.

Label one tube “(+)”. Add 10 uL of A19 glycerol stock to (+).

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Notes - you can work from glycerol stocks OR colonies.

Label the other tube “(-)”. This will be your negative control, testing if your technique is sterile.

Incubate both tubes overnight shaking at 37C / (225-250) rpm / (12-16) hr.

Perform bulk outgrowth.

Check if (-) has growth. If not, continue.

Back dilute overnight 1:250 - 1:1000 into 4x 450 mL fresh LB in 2 L baffled Erlenmeyer flasks (e.g., 1.8 mL overnight into 450 mL LB).

Incubate back diluted cultures at 37C / (225-250) rpm to mid-log phase (OD600 between 0.6 and 0.8, typically ~3 hrs).

Pellet, wash, and store cells.

Fill 1 L centrifuge bottles with culture. Balance centrifuge bottles against each other and centrifuge cultures at 16 000 rcf / 4C / 10 min.

Decant supernatant, add fresh culture, and repeat centrifugation as above, working through the remaining culture. You should end up with large pellets at the bottom of each centrifuge bottle.

Wash the pellets by resuspending (4C) NaCl (0.9%) in about 50 mL and transfer the resuspended cells to a single centrifuge bottle. Dilute to ~500 mL and re-pellet at 16 000 rcf / 4C / 10 min.

Transfer pellets by spatula into a tared bag weigh and record the mass.

Flash freeze pellet in liquid nitrogen and store at -80C.

Cell lysis:

Resuspend (2-5) g cell pellet in 25 mL of Ribosome Lysis buffer & lyse cells using 130-watt probe sonicator (probe tip diameter: 6 mm) on ice with following parameters: 50% amplitude, 15s on/ 30s off for 2 minutes on-time. The amount of energy delivered via sonication will vary depending on the amount of cells resuspended.

Clarify lysate by centrifugation at 16 000 rcf / 4C / 10 min.

Aspirate supernatant and measure volume. Add an equal volume of Salting Out buffer to adjust the concentration of ammonium sulfate to 1.5 M and mix well. Incubate at 4C / 10 min.

Remove precipitate by centrifugation at 16 000 rcf / 4C / 10 min.

Filter supernatant using a 0.22 um syringe filter and keep cold (4C).

FPLC purification

Line configuration on FPLC

Line	Buffer
A11	Ribosome Wash Buffer
B1	Ribosome Elution Buffer
A12	Water
A13	0.5 M NaOH
A14	0.1 M Acetic Acid

Set-up:

Connect the two Butyl column (5 mL) in tandem, totaling a column volume (CV) of 10 mL.

Place A1 in Ribosome Wash Buffer and B1 in Ribosome Elution Buffer. Place sample line in A2. Set the default flow rate to 4 mL / min (except for pump washes: 10 mL/min).

Equilibrate HIC column:

Perform a pump wash with Ribosome Wash Buffer (without TCEP) and equilibrate the column with 4 CV of Ribosome Wash Buffer(without TCEP).

Once you've equilibrated your columns, add TCEP to Ribosome Wash and Elution Buffer.

Load your fraction collector with 15 mL conical tubes and set the fraction volume to 5 mL.

If using a sample pump to load samples, place sample line (S1) into sample and load around 90% of sample volume onto the column. Once almost loaded, dilute the sample with Ribosome wash buffer (~5 mL) to load as much sample as possible. DO NOT allow air into the FPLC; make sure the sample line is always submerged.

Perform HIC:

Wash step 1: wash with 3 CV of Ribosome Wash Buffer to remove unbound components .

Wash step 2: wash with 5 CV of 80% Wash Buffer and 20% Ribosome Elution Buffer.

Elution: elute the product by applying 3.5 CV (35 mL) of 50% Ribosome Wash Buffer and 50% Ribosome Elution Buffer. Ensure that the fraction collector captures these fractions separately.

Wash step 3: Elute all strongly interacting contaminants with 5 CV of 100% Ribosome Elution Buffer.

Clean columns:

Place inlet into a NaOH (0.5 M) and perform pump wash. Wash the column with 3 CV NaOH (0.5M).

Place the inlet into water, perform pump wash, and then was column in 2 CV filtered Ultrapure water.

Place the inlet into AcOH (0.1 M), perform pump wash, and subsequently wash column with 3 CV AcOH (0.1 M).

Pump wash with water and wash column with 2 CV filtered MilliQ water.

Place all inlets into EtOH (20% v/v). Perform a pump wash, then wash columns with 3 CV EtOH (20% v/v). Store columns at 4C in EtOH (20% v/v) until ready for use.

Ultracentrifugation

Gently overlay recovered fractions (should correspond to second peak) onto 35 mL of Cushion Buffer in a polycarbonate ultracentrifuge bottle (70 mL).

Prepare another polycarbonate ultracentrifuge bottle as a balance. Measure 35 mL of Cushion Buffer, then add Ribosome Buffer until the balance mass is within 0.1 g of the sample bottle mass. Make sure all bottles are well balanced (Δm ≤ 0.1 g) and have no cracks!

Pellet ribosomes by ultracentrifugation at 100 000 rcf / 4C / 16 hrs. A translucent ribosome pellet will be formed at the bottom of the centrifuge bottle. It may be difficult to see.

Discard the supernatant. Carefully, wash each pellet with 0.5 mL cold ribosome buffer. Repeat this step twice.

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Notes - Don’t disturb the ribosome pellet during washing

Resuspend the clear pellets in 100 uL of Ribosome Buffer on ice using a magnetic stir bar (3 mm diameter, 10 mm length) on a magnetic stirrer set at the lowest possible speed. Collect resuspended ribosomes.

Wash tubes with an additional 50 uL of Ribosome Buffer to resuspend any remaining ribosomes.

Quality Control and Storage

Determine the ribosome concentration by measuring the absorbance at 260 nm at a 100x dilution in Ribosome Buffer. 10 units of A260 from a 100x dilution corresponds to 23 uM of undiluted solution.

Dilute to final stock of 10 uM. To adjust the concentration, dilute the ribosomes with ribosome buffer or concentrate further via centrifugation at 4000 rcf in a 100 kDa centrifugal filter at 4C.

Protein gel: dilute 10 uM sample by 4x (Add 2.5 uL of sample with 7.5 uL water) and mix with 10 uL of 4x Laemmli with BME loading buffer. Boil samples at 90C for 10 minutes and load 5 uL and 2.5 uL onto 4-20% tris-glycine gel. Run gel at 200 V / 30-45 min or until the loading dye line reaches the bottom of the gel.

Aliquot your ribosomes to reduce freeze / thaw cycles and store at -80C.

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

Cytiva HiTrap Butyl HP Instructions

HiTrap Butyl HP Instructions.pdf501.3KB

Credits

Chalie Newell, University College London
Matas Deveikis, Impeial College London