Recombinant Expression and Purification of Extracellular Domain of the Programmed CellDeath Protein Receptor

The programmed cell deathprotein 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.

We synthesized the extracellular domain of the PD-1 receptor (rPD-1) de novoby 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 Escherichia coli. 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.

Designed and synthesized construct of rPD-1 was 500 bpin 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.

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