Cell-free synthesis of membrane proteins
Materials
Preparation of mRNA
1. 5x transcription buffer (CellFree Science, Matsuyama, Japan)
2. 25 mM NTP mixture (CellFree Science, Matsuyama, Japan)
3. Ribonuclease inhibitor (80 units/µl) (CellFree Science, Matsuyama, Japan)
4. SP6 RNA polymerase (80 units/µl) (CellFree Science, Matsuyama, Japan)
Wheat germ cell-free protein synthesis
1. Wheat germ extract (WEPRO1240, 240 OD) (CellFree Science, Matsuyama, Japan)
2. Creatine kinase (Roche Diagnostics, Tokyo, Japan)
3. SUB-A-MIX (CellFree Science, Matsuyama, Japan)
4. 96-well plate (TPP Techno Plastic Products, Schaffhausen, Switzerland)
5. Plate seal (AM-111) (Tokyo Garasu Kikai, Tokyo, Japan)
6. Dialysis cup (12,000 MWCO) (Cosmo Bio, Tokyo, Japan)
7. Receptacle tube (Maruemu yohki No. 2) (Maruemu, Osaka, Japan)
8. Parafilm (Bemis Flexible Packaging, Neenah, WI)

Methods
Bilayer method
Preparation of mRNA
1. Prepare 25 µl of transcription mixture as follows: 5 µl of 5x transcription buffer, 3 µlof 25 mM NTP mixture, 2.5 µl of template plasmid (1 µg/µl), 0.25 µl of Ribonucleaseinhibitor, 0.3 µl of SP6 RNA polymerase, and 13.95 µl of Milli-Q water.
2. Incubate at 37℃ for 3-6 h.
Wheat germ cell-free protein synthesis
1. Prepare 25 µl of translation mixture on ice as follows: 6.25 µl of wheat germ extract,
0.5 µl of creatine kinase (20 mg/ml Milli-Q water), 4.7 µl of 4x SUB-A-MIX, 7.5 µl

of mRNA, 2.5 µl of liposomes, and 3.55 µl of Milli-Q water.
2. Prepare 125 µl of substrate mixture on ice as follows: 31.25 µl of 4x SUB-A-MIX
and 93.75 µl of Milli-Q water.

Figure 1

3. Transfer the 125 µl of substrate mixture to a microtiter plate.
4. Carefully pipette the 25 µl translation mixture underneath thesubstrate mixture to form bilayer (Figure 1).
5. Seal the plate to avoid evaporation.
6. Incubate the plate at 16˚C or 26˚C for 16-20 h. The typicalyield of bi-layer-mode translation is 1-5 µg/150 µl reaction
mixture.
Dialysis method
Preparation of mRNA
3. Prepare 25 µl of transcription mixture as follows: 5 µl of 5x transcription buffer, 3 µlof 25 mM NTP mixture, 2.5 µl of template plasmid (1 µg/µl), 0.25 µl of Ribonucleaseinhibitor, 0.3 µl of SP6 RNA polymerase, and 13.95 µl of Milli-Q water.
4. Incubate at 37℃ for 3-6 h.
5. Centrifuge the transcription mixture at 20,000×g for 1 min.
6. Transfer the supernatant to new centrifuge tube.
7. To the supernatant, add 3.7 µl of 7.5 M ammonium acetate and 62.5 µl of ethanol.
Mix well and incubate on ice for 15 min.
8. Centrifuge the mixture at 20,000×g for 20 min at 4˚C.
9. Discard the supernatant, and rinse the pellet with 500 µl of 70% ethanol.
10. Dry the pellet and dissolve the dried pellet in 12.5 µl of Milli-Q water.
Wheat germ cell-free protein synthesis
1. Prepare 50 µl of translation mixture on ice as follows: 12.5 µl of wheat germ extract,
1 µl of 20 mg/ml creatine kinase, 9.4 µl of 4× SUB-A-MIX, 12.5 µl of mRNA, 5 µlof liposomes, and 9.6 µl of Milli-Q water.
2. Prepare 800 µl of substrate mixture on ice according as follows: 200 µl of 4× SUBA-MIX and 600 µl of Milli-Q water.
3. Transfer the 50 µl translation mixture into a dialysis cup.
4. Transfer 800 µl of the substrate mixture into a receptacle tube.

5. Place the dialysis cup containing the translation mixture into the receptacle tube.
6. Seal the connected portion with parafilm to avoid evaporation.
7. Incubate the reaction mixture at 16˚C or 26˚C for 16-20 h. The typical yield of dialysis
method is 5-20 µg/50 µl translation mixture.
Bilayer-dialysis method
Preparation of mRNA
1. Prepare 25 µl of transcription mixture as follows: 5 µl of 5x transcription buffer, 3 µlof 25 mM NTP mixture, 2.5 µl of template plasmid (1 µg/µl), 0.25 µl of Ribonucleaseinhibitor, 0.3 µl of SP6 RNA polymerase, and 13.95 µl of Milli-Q water.
2. Incubate at 37℃ for 3-6 h.
3. Centrifuge the transcription mixture at 20,000×g for 1 min.
4. Transfer the supernatant to new centrifuge tube.
5. To the supernatant, add 3.7 µl of 7.5 M ammonium acetate and 62.5 µl of ethanol.
Mix well and incubate on ice for 15 min.
6. Centrifuge the mixture at 20,000×g for 20 min at 4˚C.
7. Discard the supernatant, and rinse the pellet with 500 µl of 70% ethanol.
8. Dry the pellet and dissolve the dried pellet in 12.5 µl of Milli-Q water.
Wheat germ cell-free protein synthesis
1. Prepare 50 µl of translation mixture on ice as follows: 12.5 µl of wheat germ extract,
1 µl of 20 mg/ml creatine kinase, 9.4 µl of 4× SUB-A-MIX, 12.5 µl of mRNA, 5 µlof liposomes, and 9.6 µl of Milli-Q water.
2. Prepare 1,200 µl of substrate mixture on ice according as follows: 300 µl of 4× SUBA-MIX and 900 µl of Milli-Q water.
3. Transfer the 150 µl the substrate mixture into a dialysis cup.
4. Transfer the 50 µl translation mixture underneath the substrate mixture in the dialysis
cup to form bilayer.
5. Transfer 800 µl of the substrate mixture into a receptacle tube.
6. Place the dialysis cup into the receptacle tube.
7. Seal the connected portion with parafilm to avoid evaporation.
8. Incubate the reaction mixture at 16˚C or 26˚C for 16-20 h.