Expression,Purification and Activity Determination of PD-133-150 and PD-L119-239 Proteins
10.13865/j.cnki.cjbmb.2025.06.1220
- VernacularTitle:PD-133-150和PD-L119-239蛋白质的表达、纯化及活性检测
- Author:
Xin-Rong YU
1
;
Xiao-Hong QIN
1
;
Li-Zhi MI
1
Author Information
1. 天津大学医学部生命科学学院结构与分子生物学研究所,天津 300072
- Publication Type:Journal Article
- Keywords:
programmed cell death protein 1(PD-1);
programmed cell death 1 ligand 1(PD-L1);
golden gate assembly;
eukaryotic expression;
activity assay
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(8):1193-1203
- CountryChina
- Language:Chinese
-
Abstract:
Programmed cell death protein 1(PD-1)and its ligand,programmed cell death 1 ligand 1(PD-L1),represent a pair of prototypical immune checkpoint that plays a critical role in tumor immune evasion.However,the development of targeted therapeutics against these two proteins is limited by their low levels and high glycosylation modifications in eukaryotic cells.In this study,we designed two func-tional but truncated variants of PD-1(PD-133-150)and PD-L1(PD-L1 19-239);and subcloned them into eukaryotic expression vectors using golden gate assembly technology.Using these vectors,we achieved high level yields of these two proteins in transiently-transfected HEK-293T cells.After one-step affinity purification,the yields of PD-133-150 and PD-L119 239 proteins reached 5 mg and 3 mg per liter of cell cul-ture medium,with over 95％purity.Using biolayer interferometry and flow cytometry analysis,we deter-mined the binding kinetics,equilibrium constants,and the cellular binding activities of these proteins.Compared with the PD-1 extracellular domain expressed in insect or E.coli cells,the PD-133-150 purified from HEK-293T cells has a 24-fold and a 50-fold increase in its binding affinity to PD-L1.In addition,the dissociation rate of the binding decreased to less than l/400th of the original rate.Thus,we speculate that N-glycosylation could modulate the PD-1/PD-L1 interactions.Together,we established an effective eukaryotic expression and purification platform for functional characterization of PD-133150/PD-L119239 in-teractions,thereby providing high-quality molecular tools for PD-1/PD-L1 antibody screening and im-mune checkpoint research.
