Reports of Biochemistry

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Zhansaya A, Kanatbek M, Kanat T, Bakhytkali I, Darkhan K, Arman K, et al . Recombinant Expression and Purification of Extracellular Domain of the Programmed CellDeath Protein Receptor. rbmb.net 2020; 8 (4) :347-357
URL: http://rbmb.net/article-1-391-en.html

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