Production of Olfactory Receptors Using Commercial E. coli Cell-free Systems - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 116

Table 7.1 Solubility and maximum yields of GPCRs produced using cell-free in vitro translation
in the presence of Brij-35
GPCR % solubility Yield (mg) a GPCR % Solubility Yield (mg) a

Olfr226 86 ± 8 3.7 hOR17-209 88 ± 4 2.5

mOR33-1 85 ± 2 5.9 hOR17-210 91 ± 2 4.5

mOR103-15 90 ± 4 4.5 hFPR3 83 ± 5 5.5

2.4 b

mOR106-13 86 ± 13 hTAAR5 90 ± 1 4.5

mOR174-4 89 ± 2 2 hVN1R1 88 ± 0.1 0.4

mOR174-9 86 ± 3 6 hVN1R5 85 ± 2 1 b

mOR175-1 81 ± 8 2.5
a Milligrams of receptor that could be produced in a 10 ml cell-free reaction. These yields were
calculated from smaller batches of protein purified using immunoaffinity chromatography. Experi-
ments showed that up to 1 mg/ml of protein could be produced, but that up to half could be lost
during the purification process. The yields were determined by spectrophotometer readings
b These yields were calculated by comparing the intensities of the receptor samples against a
sample with a known concentration

Aliquots can be made for larger volumes of buffer. The specific buffer varies
with each kit, but contains all essential amino acids, RNA polymerases, ribo-
somes, elongation factors, etc.
3. Sterile, DNase-free and RNase-free water
4. Non-ionic Detergent Brij-35 [2 % (10X) or 10 % (50X) concentration]
5. Olfactory receptor gene ligated into the pIVex2.3d plasmid vector (Life
Technologies)

7.2.2 Cell-Free Protein Production

1. Thaw the E. coli lysate, reaction buffer, and DNA on ice.
2. Add 175 µl of the E. coli lysate to a sterile, DNAse-free, RNAse-free Eppen-
dorf tube Add the plasmid to the lysate so that the final DNA concentration is
1 µg/100 µl.
3. Add DNAase-free and RNAse-free water so that the final volume of the cell-
free reaction will be 500 µl.
4. Add Brij-35 to a final concentration of 0.2 % w/v.
5. Add 200 µl of the reaction buffer, and mix thoroughly with a pipette.
6. Briefly (~ 5 s) spin down the Eppendorf tube.
7. Place the cell-free reaction in an Eppendorf rack in a shaking incubator at 33 °C
and 250 rpm for 1 h.
8. After the reaction is complete, spin it down in a microcentrifuge for 5 min at
10,000 rpm.
9. Carefully transfer the supernatant to a fresh tube without disturbing the pellet.
The supernatant contains solubilized receptor.
10. The synthesized receptors can be purified or run on an SDS-PAGE gel immedi-
ately, or stored at − 20 °C for longer periods of time.