Expression and purification of soluble human programmed death-1 in Escherichia coli.
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.
Xu
Lihui
L
Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Liu
Yi
Y
He
Xianhui
X
D015703:Antigens, CD / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D060890:B7-H1 Antigen; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D061026:Programmed Cell Death 1 Receptor; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D015703:Antigens, CD; D051017:Apoptosis Regulatory Proteins; D060890:B7-H1 Antigen; C423236:CD274 protein, human; C105992:PDCD1 protein, human; D061026:Programmed Cell Death 1 Receptor; D011993:Recombinant Fusion Proteins