recombinant expression and purification of extracellular domain of the programmed celldeath protein receptor

Background: 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.methods: 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.results: 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.conclusions: synthesized de novo rpd-1 may have potential therapeutic applications in enhancing antitumor immune responses