Recombinant Expression and Purification of Extracellular Domain of the Programmed Cell Death Protein Receptor.

BACKGROUND

The programmed cell death protein 1 (PD-1), which is a member of the CD28 receptor family, can negatively regulate antitumor immune responses by interacting with its ligands, PD-L1 or PD-L2. The PD-1-PD-L1 signaling pathway is a checkpoint mechanism that plays essential roles in downregulating immune responses in cancerous tissues. Thus, blocking this signaling pathway leads to enhanced antitumor immunity, potentially preventing tumor progression.

METHODS

We synthesized the extracellular domain of the PD-1 receptor (rPD-1) de novo by using a two-step polymerase chain reaction and the Phusion® DNA polymerase. The synthesized gene was cloned into the pET28 expression plasmid and transformed into competent . Purification of rPD-1 was performed by metal-affinity chromatography, using a HisTrap column. Purified rPD-1 was characterized by western blotting and mass spectrometry using the SwissProt database and the Mascot program.

RESULTS

Designed and synthesized construct of rPD-1 was 500 bp in size. Analysis of the electrophoresis data of purified rPD-1 showed the presence of a protein with a molecular mass of 21 kDa. Mass spectrometry data using the SwissProt database and the Mascot program outputted the highest-scoring sequence to correspond to rPD-1.

CONCLUSION

Synthesized de novo rPD-1 may have potential therapeutic applications in enhancing antitumor immune responses.