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Published on: September 28, 2018
Recombinant Expression and Purification of Extracellular Domain of the Programmed Cell Death Protein Receptor
Adish Zhansaya1,2, Mukantayev Kanatbek1, Tursunov Kanat1
1National Center for Biotechnology, Kurgalzhyn road, 13/5, Astana, 010000, Kazakhstan.
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 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 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.
Insights
Researchers synthesized the programmed cell death protein 1 (PD-1) receptor de novo. This synthesized rPD-1 may enhance antitumor immune responses for potential cancer therapies.
Area of Science:
- Immunology
- Molecular Biology
- Protein Engineering
Background:
- Programmed cell death protein 1 (PD-1) negatively regulates antitumor immunity by interacting with PD-L1/PD-L2.
- The PD-1-PD-L1 pathway is a critical immune checkpoint in cancer, downregulating anti-tumor responses.
- Blocking this pathway can enhance immune responses against tumors, potentially halting progression.
Purpose of the Study:
- To synthesize the extracellular domain of the PD-1 receptor (rPD-1) de novo.
- To characterize the synthesized rPD-1 for potential therapeutic applications.
Main Methods:
- De novo gene synthesis of rPD-1 using polymerase chain reaction and Phusion® DNA polymerase.
- Cloning into pET28 expression plasmid and transformation into *Escherichia coli*.
- Purification via metal-affinity chromatography and characterization by western blotting and mass spectrometry.
Main Results:
- Successfully synthesized a 500 bp construct of rPD-1.
- Purified rPD-1 exhibited a molecular mass of 21 kDa via electrophoresis.
- Mass spectrometry confirmed the identity of the synthesized protein as rPD-1.
Conclusions:
- De novo synthesized rPD-1 shows promise for therapeutic development.
- This engineered protein could potentially be used to enhance anti-tumor immune responses.
