Establishment of PD-1 Flag-tagged constructs

Establishment of PD-1 Flag-tagged constructs

1. The lentiviral expression constructs were based on the pCDH-EF1-MCS-T2A-copGFP expression plasmid (System Biosciences) 

2. The restriction enzymes XbaI and EcoRI were used in order to insert the respective PD-1 Flag-tagged sequence into the pCDH-EF1-MCS-T2A-copGFP expression plasmid.

3. The amino acid sequences of PD-1 WT (protein sequence in UniProt Q15116) or PD-L1 WTf (protein sequence in UniProt Q9NZQ7) from the Uniprot website were used. The amino acid sequence of PD-1 WT was then changed to PD-1 Y223F or PD1 Y248F, respectively. 

4. N-terminal of the PD-1 WT sequence, the sequence for the XbaI restriction site (GGCGCC) and the Kozak sequence were added (GCCGCCACC). C-Terminal of the respective PD-1 sequence a linker sequence (GGGGS), the single Flag-Tag sequence (DYKDDDDK) and the sequence of the EcoRI restriction site (GAATTC) were added.

5. The respective constructs as amnio acid sequence were separately (PD-1 WT Flag, PD-1 Y223F Flag, PD-1 Y248F Flag) codon-optimized for expression in Homo sapiens (human) using the Eurofins website and transformed into the DNA sequence.

6. The obtained codon-optimized sequences for expression in humans were then assembled as gBlocks and ordered from Integrated DNA Technologies (IdT DNA).

7. The gBlock constructs were dissolved in 100 μl Elution buffer AE (5 mM Tris HCl pH 8.5) to obtain a final conc of 10 ng/μl.

8. The gBlock constructs were amplified using the Q5 Polymerase and suitable primers.

9. The obtained PCR products were then cloned into the pCDH-EF1-MCS-T2A-copGFP expression plasmid (System Biosciences) using the Xba I and EcoRI restriction sites.