| Products/Services Used | Details | Operation |
|---|---|---|
| DNA Sequencing | Cloning primers and synthetic genes encoding 33- 150 residues of hPD-1 (NCBI: NM_005018.2) and 19- 229 residues of human PD-L1 (GenBank: AY254342.1) were ordered from GenScript (Nanjing, China) for the expression in E. coli. The hPD-1 residue 93 Cys was mutated to Ser to remove a free thiol from the bacterial expressed protein for improving refolding. The PCR products of hPD-1 and hPD-L1 genes were digested with Nco I and Not I endonuclease (NEB, Ipswich, MA, USA), and ligated into the pET28a vector (Novagen, Madison, WI, USA) which had a biotin tag gene for encoding tagged proteins and predigested by the identical restriction enzymes. The genes of hPD-L1 variants obtained from hPD-L1 phage libraries were amplified by PCR from the pGZ196 vector (in house construct), and cloned into the pET28a vector. The ligated products were transformed routinely into the competent E. coli strain BL21 (DE3) (Vazyme, Nanjing, China). Successful clones were confirmed with DNA sequencing | Get A Quote |
The activated T cells can be suppressed by programed death-1 (PD-1) axis through low affinity interaction between PD-1 and PD-ligand 1 (PD-L1) in solution or on antigen presenting cells. In clinic, the concentration of soluble PD-L1 in peripheral blood negatively correlates with cancer prognosis. However, there is little information about the relation between the affinity of PD-1/PD-L1 interaction and the suppressive capacity of PD-1 axis. In this study, we analyzed inhibitory roles of high affinity soluble human PD-L1 (hPD-L1) variants, which were generated with directed molecular evolution. Resultant two clones L3C7-hPD-L1 and L3B3-hPD-L1 showed over 20 folds greater affinity than that of native hPD-L1. We fo... More
