Application Note: Rapid Cell-Free Synthesis and Purification of a Red Fluorescent Protein

Background

Monomeric Red Fluorescent Protein (mRFP1) was created by Campbell et al. in 2002. It is a constitutively fluorescent protein derived from the Discosoma species of mushroom anemone, a soft coral, and has a somewhat slow maturation time of about an hour. Due to its compact size and brilliant colour, it has been used in a host of synthetic biology applications.

Here, we demonstrate the synthesis and purification of the C-terminally His-tagged mRFP1 protein from a pJLA1-based plasmid using cell-free protein expression reagents and cartridges. The reactions were carried out at two different scales: 1 mL and 300 µL. It is important to highlight that the purification components are not linearly scaled between different reaction sizes – if you require help with your specific experimental designs, please reach out to us.

 

Cell-Free Synthesis of mRFP1

DNA was prepared using Epoch DEAE maxi columns and eluted in nuclease free water. The two reactions were set up as follows:

Resuspension Buffer

DNA Template

(782 ng/µL)

Solution A

Solution B

Cell Extract

Final Volume

1 mL Reaction

370.50

37.08

114.63

164.46

343.33

1030.00

300 µL Reaction

118.48

12.10

36.73

52.69

110.00

330.00

 

The components were added in in the same order from left to right for each reaction volume. Components were added to a 1.5 mL Eppendorf tube and vortexed briefly prior to being transferred to the cell-free protein expression cartridges. The 1 mL reaction was pipetted into a 300 - 1000 μL Feather™ Cell-Free Expression Bioreactor Spin-Column. The 300 µL reaction was pipetted into a 100 - 300 μL Feather™ Cell-Free Expression Bioreactor Spin-Column. The cartridges were placed back inside of their corresponding tubes, the tubes were capped and incubated at 26°C for approximately 16 hours. The brilliant red colour of mRFP1 could clearly be identified through the body of the cartridge after about an hour. 15 µL of each reaction was also pipetted into a 1.5 mL Eppendorf Tube and incubated at 26°C for approximately 16 hours, providing an initial sample for SDS-PAGE analysis.

mRFP Synthesis

 

Nickel-NTA Purification of His-Tagged mRFP1

The mRFP1 construct used in this example contains a C-terminal octa-histidine tag. As such, nickel NTA purification was used. The buffer formulations in this example were as follows:

Purification Step

Buffer Components

Binding

250 mM NaCl, 5% glycerol, 50 mM HEPES (pH 7.5), 0.5 mM TCEP (add immediately before use)

Wash

250 mM NaCl, 20 mM imidazole, 5% glycerol, 50 mM HEPES (pH 7.5), 0.5 mM TCEP (add immediately before use)

Elution

250 mM NaCl, 250 mM imidazole, 5% glycerol, 50 mM HEPES (pH 7.5), 0.5 mM TCEP (add immediately before use)

 

We adjusted purification buffer volumes and the amount of bead slurry used for each reaction volume. The table below outlines the specific volumes used in each case:

Step

300 µL Reaction

1 mL Reaction

Volume of bead slurry

80 µL

200 µL

Volume of water/binding buffer used for washing and equilibrating the beads

200 µL

500 µL

Volume of binding buffer used for resuspending the beads

200 µL

400 µL

Volume of wash buffer

2 x 400 µL

2 x 1000 µL

Elution buffer

2 x 150 µL

2 x 500 µL

 

     1. Preparing the resin:

    • Resin slurry was taken out of the fridge and centrifuged at 500 x g for 2 minutes at 4°C. The liquid portion was then removed, taking care not to disturb the beads.
    • The beads were washed with molecular grade water, and centrifuged at 500 x g for 2 minutes at 4°C. The liquid portion was then removed, taking care not to disturb the beads.
    • The beads were equilibrated with binding buffer, and centrifuged at 500 x g for 2 minutes at 4°C. The liquid portion was then removed, taking care not to disturb the beads.
    • The beads were then resuspended in binding buffer.
Note: the buffers were chilled to 4°C prior to use and all the following steps were carried out on ice.
 
  1. Resuspended beads were pipetted directly into the cartridges containing the cell-free reactions.
  2. The caps were placed tightly back on the tubes, and the contents were inverted several times. Inversion was repeated periodically for 15 minutes.
  3. The spin columns were centrifuged at 4000 x g for 2 minutes at 4°C. Flow-through was collected for SDS-PAGE analysis.
    mRFP flow-through
  4. Wash buffer was pipetted into each tube, and the tubes were gently inverted several times. The spin columns were then centrifuged at 4000 x g for 2 minutes at 4°C. The used wash buffer was kept for SDS-PAGE analysis.
    mRFP Wash
  5. The wash step was repeated for each spin column.
  6. The spin columns were then placed into fresh tubes, elution buffer was added into each. The beads were incubated with the elution buffer for 5 minutes, and were periodically gently inverted. The spin columns were then centrifuged at 4000 x g for 2 minutes at 4°C. The eluate was collected for use and for SDS-PAGE analysis.
    mRFP Elution
  7. The elution step was then repeated for a second round.

 

SDS-PAGE Analysis

The collected samples were analyzed on a 15% SDS-PAGE gel run at 200 V for 60 minutes (2 µL of each sample were loaded). The image below shows the results for the 1 mL reaction. The tube marked as initial contains the 15 µL sample of the reaction.

 

Conclusion

Here, we have demonstrated the synthesis and purification of mRFP1 using cell-free protein expression reagents and cartridges in under 24 hours. These results showcase the ease of use and power of cell-free protein synthesis for small-scale protein production.

 

Acknowledgements

Experiments presented here were carried out by Taylor Sheahan.