Recombinant PD-L1 and PD-1 Constructs

Constructs encoding the extracellular regions of human PD-L1 D1D2 (18–237), PD-L D1 (18–134), and PD-1 (33–150) with a C-terminal avitag (GGLNDIFEAQKIEWHE) were synthesized and cloned into pET28a by GenScript. A C-terminal avitag construct of PD-L1 D1D2 (18–237) was produced by mutagenesis (https://openwetware.org/wiki/%27Round-the-horn_site-directed_mutagenesis, accessed February 2, 2022) (58 (link)). PD-L1 D2 (130–239) was cloned into the pLEICS-05 vector by PROTEX (University of Leicester). PD-L1 and PD-1 constructs were expressed and refolded as previously reported (6 (link)). Proteins were purified using a Superdex 75 26/600 gel filtration column (GE Healthcare) equilibrated in 25 mM KH₂PO₄, pH 7.4, 25 mM NaCl, and 10 μM EDTA. Isotopically labeled PD-L1 D1 was expressed in modified Spizizen’s minimal media (59 (link), 60 (link)), incorporating ¹⁵N-NH₄Cl and ¹³C-glucose, and was refolded and purified as aforementioned.