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Expression and purification of soluble human programmed death-1 in Escherichia coli
1Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Abstract:
Programmed death-1 (PD-1), a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands (PD-L1 and/or PD-L2). PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag (designated as sPD-1) was expressed as inclusion bodies in Escherichia coli. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.
Insights
Researchers developed soluble Programmed death-1 (PD-1) from E. coli. This soluble PD-1 effectively binds to PD-L1, showing potential for enhancing anti-viral and anti-tumoral immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Programmed death-1 (PD-1) negatively regulates T cell receptor signaling via ligands PD-L1/PD-L2.
- The PD-1/PD-L1 pathway is crucial in adaptive immune responses and is a target for enhancing anti-viral and anti-tumoral immunity.
Purpose of the Study:
- To express and characterize soluble human PD-1 (sPD-1) from prokaryotic cells.
- To assess the binding activity of refolded sPD-1 with its ligand PD-L1.
Main Methods:
- Expression of extracellular human PD-1 with a His-tag in E. coli.
- On-column refolding and purification using immobilized metal affinity chromatography.
- Characterization by Western blotting and Scatchard plot analysis for binding affinity.
Main Results:
- Soluble PD-1 (sPD-1) was successfully expressed as inclusion bodies and refolded.
- Purified sPD-1 demonstrated high purity and specific binding activity to PD-L1.
- The dissociation constant (Kd) for sPD-1 and PD-L1 binding was determined to be 0.43 nmol/L.
Conclusions:
- Refolded sPD-1 from prokaryotic expression systems can be produced with high purity and specific ligand-binding activity.
- This soluble PD-1 has potential therapeutic applications for enhancing immune responses against viral infections and malignancies.
