Scale-up Expression and Purification

Scale-up expression and purification

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General information

The eProtein Discovery™ Scale-up protocol allows the in-tube protein expression of a specific eGene™ construct (DNA) using E. coli derived Cell-free Protein Synthesis (CFPS) reagents and additives.

The selection of the optimal eGene™ / Cell-Free Blend combination (eRecipe) is determined from the expression and purification screens performed on the eProtein Discovery instrument.

Depending on the protein of interest, the expected yield is as predicted from the experiment on the eProtein Discovery instrument.

The scale-up reaction volume in this guide is 200 µL. However, larger scale-up reactions, for example 1 mL, can also be achieved simply by adapting proportionally the volumes of the reagents and the Strep Beads.

The whole scale-up experiment takes less than 24 hours, as summarized in Table 1

Protocol Step	Reagents and equipment required	Time
Expression reaction setup	* Cell Free Core Reagent	30 min
	* Scale-up Additives (list in Table 2)
	* 5 nM eGene™ construct (DNA)
Protein Expression reaction	* Incubator, constant temperature of +29°C	15-18 hours (overnight)
	* No agitation required
Protein Purification	* Magnetic particle separator

The Scale-up kit NC3011 contains the Cell-free Core reagent, the purification Strep beads and the Wash and Elution buffers. The Scale-up Additives Kit NC3005 contains the additives listed in Table 2. The components of NC3011 and NC3005 are supplied in a Nuclera branded box with a grey stripe on the label.

Additive name	Additive Description	Additive Characteristics
Additive Buffer	HEPES buffer pH 7.5 and surfactant	CFPS reaction buffer
PDI + GSSG Mix	Protein disulfide isomerase and oxidized glutathione	Chaperone and redox modification to oxidizing environment to support disulfide bond formation
TrxB1	Thioredoxin reductase	Protects proteins from oxidative aggregation and inactivation and acts as a reductase in redox regulation
DnaK Mix	Chaperone DnaK mix	Chaperone mix to support folding and prevent aggregation
Zinc chloride	Zinc chloride solution	Cofactor that can be required for folding, stability, or activity
Calcium chloride	Calcium chloride solution	Cofactor that can be required for compaction, folding, stabilization, or activity
Manganese chloride	Manganese chloride solution	Cofactor for metalloenzymes for structure and activity
Cofactor Mix	Mix of NAD, acetyl-CoA,FAD, SAM, and PLP	Cofactors that assist in folding, stability and activity
GSSG	Oxidized glutathione	Redox modification to oxidizing environment
3C protease	3C protease solution	Protease to cleave off the N-terminal solubility tag at the specific amino-acid sequence

Scale up bundles content

Scale-up kit (NC3011)

Strep beads and Scale-up reagents must be used within the expiration date stated on the kit box.

Scale-up kit Strep Beads NC3011-1	Cap color	Quantity	Storage instruction
Strep Beads	Orange	5x400 µL	+4°C

Scale-up kit Strep Beads NC3011-2	Cap color	Quantity	Storage instruction
Cell-free Core Reagent	Purple	5x160 µL	-80°C
Wash Buffer	White	5x3 mL	-80°C
Elution Buffer	Blue	5x300 µL	-80°C

Scale-up additives (NC3005)

Scale-up additives must be used before the expiration date indicated on the box.

Scale-up kit Strep Beads NC3011-2	Cap color	Quantity	Storage instruction
PDI/GSSG mix*	Green	1x175 µL	-80°C
TRXB1*	Green	1x175 µL	-80°C
DNAk mix*	Green	1x175 µL	-80°C
Zinc chloride	Green	1x175 µL	-80°C
Calcium chloride	Green	1x175 µL	-80°C
Manganese chloride	Green	1x175 µL	-80°C
Cofactor mix*	Green	1x175 µL	-80°C
GSSG*	Green	1x175 µL	-80°C
3C protease*	Green	1x175 µL	-80°C

* Single use reagent that cannot be freeze/thawed multiple times

User supplied reagents (not included in the kit)

• 5 nM eGene construct (DNA) generated using the Nuclera eGene Prep kit NC3009 or NC3008

User supplied equipment

• Incubator (capable of maintaining a constant temperature of 29ºC)
• Magnetic particle separator (compatible with 1.5 mL microcentrifuge tubes)
• P1000, P200 pipettes and tips
• Vortexer
• Microcentrifuge
• 1.5 mL microcentrifuge tubes
• A tube rotator / agitator

Scale-up expression and purification workflow

Step 1: Cell-free Protein Synthesis (CFPS) reaction setup

1. Take the Cell-free Core Reagents aliquot, the two selected Scale-up Additives and the eGene construct from the freezer, and allow to thaw on ice. This will take approximately 5 minutes. Once thawed, keep the reagents on ice.
2. Centrifuge the Cell-free Core Reagent aliquot at 1000 rpm for 2 seconds and return to ice.
3. In a 1.5 mL microcentrifuge tube, set up the CFPS expression reaction according to Table 3

CFPS expression

Reagents	Volume
Cell Free Reagent	120 µL	600 µL
Selected Additive 1	15 µL	75µL
Selected Additive 2	15 µL	75µL
5 nM eGene DNA construct	50 µL	250µL
Total reaction	200 µL	1 mL

Table 3: CFPS expression reaction set-up, 200 µL or 1 mL.

Note: it is recommended to run a 20 µL no-DNA negative control in parallel. In this case, substitute the 5 nM eGene construct with sterile water. Loading 3 µL of the negative control allows one to determine where the protein of interest is on the SDS-PAGE gel.

4. Vortex reaction tubes for 2 seconds to mix.
5. Centrifuge at 1000 rpm for 10 seconds.
6. Place samples in a tube rack and incubate the reaction mixture at 29ºC overnight (15-18 hours) in a temperature controlled incubator.

Note: there is no requirement to agitate the samples during incubation.

Step 2: Purification procedure

Note: the volumes and number of vials indicated in this procedure are for 200 µL CFPS reaction solutions.

Note: for optimal purification, it is recommended not to use CFPS reaction solutions larger than 500 µL. For example, a 1 mL CFPS reaction should be split into two 500 µL tubes before purification. Refer to the last column of Table 4 for volumes used for a 1 mL CFPS reaction.

Note: the Wash Buffer and Elution Buffer contain a non-ionic detergent, to keep the purification conditions the same as on the eProtein cartridge. If the protein is required without detergents then please contact the Nuclera Technical Support team.

1. Take one vial of Wash Buffer (3 mL) and one vial of Elution Buffer (300 µL) from the freezer, and allow them to thaw at room temperature. This will take approximately 20 minutes.
2. Vortex buffers for 5 seconds to homogenize.
3. Take one vial of 400 µL Strep beads supplied in the kit from the fridge.
4. Prepare the Strep Purification Beads:

• Give the vial(s) of Strep Beads a quick spin in a microcentrifuge to pellet the beads
• Pipette up and down to fully resuspend the beads.
• Transfer the beads to a 1.5 mL microcentrifuge tube.
• Place the tube(s) of Strep Beads for 1 minute on a magnetic particle collector to pellet the beads.
• Aspirate the storage buffer supernatant and discard.
• Remove the tube from the collector and resuspend the Strep Bead pellet with 400 µL Wash Buffer by pipetting up and down repeatedly.
• Repeat steps c, d and e for a total of 2 washes.
• Pellet the Strep Beads on the magnetic particle collector, aspirate and discard the supernatant.
• Remove the tube from the collector and resuspend the Strep Bead pellet with 400 µL Wash Buffer to create a working solution ready to use (5% v/v).

5. Remove the 200 µL CFPS reaction tube prepared in step 1 from the 29°C incubator.
6. Give the CFPS a quick spin in a microcentrifuge.
7. Remove and reserve 3 µL of the CFPS reaction to run on a SDS-PAGE gel later (Label: Crude CFPS).
8. Pipette up and down three times the 400 µL Strep Bead suspension prepared in step 6 and place the tube on a magnetic particle collector for at least one minute to capture the beads.
9. Aspirate the supernatant and discard.
10. Remove the tube from the magnetic particle collector, centrifuge briefly to collect any residual liquid at the bottom of the tube, and then return the tube to the magnetic particle collector.
11. If there is any significant liquid remaining over the pellet, remove using a P10 pipette. Note: be careful to not remove any beads.
12. Pipette and transfer the CFPS reaction solution to the tube containing the Strep Beads.
13. Pipette up and down 10 times to resuspend the beads and incubate the suspension for 30 minutes at room temperature with agitation using a tube rotator or shaker at about 400 rpm. The beads should be suspended throughout the 30 min to ensure an optimal binding capacity.
14. After 30 minutes of incubation, place the tube on a magnetic particle separator and pellet the Strep Beads for 1 minute.
15. Aspirate and transfer supernatant to a new microcentrifuge tube. This supernatant contains all of the unbound, contaminating proteins from the CFPS reaction, along with any unpurifiable target protein. Retain the supernatant to run on an SDS-PAGE gel.
16. Remove the tube from the collector and resuspend the purification bead pellet in 400 µL of Wash Buffer. Pipette up and down 5 times to mix.
17. Place the tube on a magnetic particle separator and pellet the Strep Beads for 1 minute.
18. Aspirate the supernatant and discard. Retain the bead pellet and carry forward to the next step.
19. Repeat twice steps 17-19 for a total of 3 washes.
20. Resuspend the beads in 250 µL Elution Buffer and place the tube on a tube rotator or shaker for 10 mins to elute the protein. Note: For proteins predicted to be expressed at 125 µg/mL or less, we recommend to use only 125 µL elution buffer.
21. Place the tube on a magnetic particle separator and pellet the Strep Beads for 60 seconds.
22. Aspirate and transfer supernatant into a new microcentrifuge tube (label aspirate: Purified). This tube contains the purified protein and can be stored for analysis and downstream applications.
23. Discard the Strep Bead pellet.

The expression and purification steps are summarized in Table 4.

Component	Volume
CFPS reaction	200 µL	1 mL
Prepared Strep Beads (5% v/v in Wash Buffer)	400 µL	2 x 1 mL*
Wash 1	400 µL	2 x 1 mL*
Wash 2	400 µL	2 x 1 mL*
Wash 3	400 µL	2 x 1 mL*
Elution Buffer	250 or 125 µL	2 x 625* µL or 2 x 312.5* µL

Table 4: Scale-up Kit purification summary. *When purifying CFPS reactions larger than 500 µL it is recommended to split the volume in two. Proportionally calculate the total volume required for the process and divide it into equivalent volume smaller or equal to 500 µL.

Prepare samples for SDS page gel

This section aims to give a guideline to run a commercial 15 combs SDS page gel.

For the SDS page gel you will need 4 tubes

	Sample	Lab Grade Water	3.6x Loading reducing dye
Eluted Protein	3 µL	3.3 µL	5.7 µL
Core + NFW (negative control)	3 µL	19 µL	9 µL
Crude CFPS	3 µL	19 µL	9 µL

Standards
To have a semiquantitative assay, you can prepare and run BSA standards alongside your samples.
Dilute your BSA sample to 1 mg/mL and make dilution as outlined in the table below to prepare your 3 standards (A, B and C)

	Required Stocl	Lab Grade Water	3.6x Loading reducing dye
Standard A	BSA 1 mg/mL	24 µL	176 µL
Standard B	Standard A	50 µL	150 µL
Standard C	Standard B	50 µL	150 µL

Load the SDS PAGE gel
Load on the stain-free protein gel, 4-15% (15 well):

• 4 µL pre-stained protein ladder
• 4 µL eluted scaled-up protein sample
• 8 µL eluted scaled-up protein sample
• 4 µL No-DNA negative expression control
• 4 µL positive expression control
• 4 µL BSA standard A
• 4 µL BSA standard B
• 4 µL BSA standard C

Run at 200 V for 40 minutes

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