Expression of T1R2-T1R3
To synthesis human sweet receptor gene T1R2-T1R3, we firstly got its sequence from NCBI and used Snap Gene to optimize the sequence. Then we synthesized the target gene by using OLIGO (oligo design software).
To begin with, we designed 45 pairs of primers for each sweet receptor and each primer had a 15bp overlap region at each end. We divided the primers into 3 groups named block A, B and C, and mixed the primers of each group respectively with a final concentration of 10μM. We got 6 ideal fragments successfully after PCR and purification.
To verify whether the heterodimer had been expressed and located at the proper position successfully, we set different color proteins as tags to each sweet taste receptor. We chose blue, yellow and green fluorescent tags separately. For the primer design, we added another 20bp of N-terminal of each sweet taste receptor as the overlap region. The fluorescent tags were linked to each sweet taste receptor by OE-PCR.
Another way for verification was to add different epitope tags to each sweet taste receptor.
However, according to the protein expression process, adding color proteins on the N-terminal of the target protein may not provide enough time for color protein to fold at its functional state before being secreted. So we plan to use His tag along with antibody detection.
In order to express the human receptor T1R2-T1R3, we chose Saccharomyces cerevisiae strain CEN.PK2-1C as the host and pESC-Leu as the shuttle vector. We selected two restriction sites, BamHI and SalI, for constructing gene T1R2; SpeI and NotI for T1R3. And to confirm the expression and location of the protein, tag Myc and tag His were linked to T1R2 and T1R3 respectively.
The digested DNA fragments were cloned into plasmid pESC-Leu. Then we transformed the recombinant plasmid into the E.coli BMTop10 and screened positive clones by colony-PCR.
The recombinant plasmid in E.coli was extracted and transformed by LiAc transformation method into CEN.PK2-1C which contains the Pfus-mRFP-CYC1t(G418 resistance vector) gene circuit, a detection device.
With the help of auxotrophic selection marker Leu in pESC-Leu, the colony was chosen by the Leucine-defect SD medium containing 0.3% G418.
In this way, we got the recombinant Saccharomyces cerevisiae with sweet taste receptor and detection device successfully.
T1R2-T1R3 with His tag and Myc tag was designed to confirm the expression and location of T1R2-T1R3 in the CEN.PK2-1C, which was tested through the immunofluorescence technology.
Then we used the minimal induction medium to induce the Gal1/Gal10 promoter. After protein expressed, we incubated the primary antibody for His tag and Myc tag, then added second antibody for displaying the expression and location of receptors in yeast cells.