Periplasmic extractions of small-scale cultures

PEs were produced as described [53]. Briefly, E.coli WK6 cells were transformed with Nb-coding pHEN4 plasmids and cultured in 10 mL Terrific broth (TB) containing 100 μg/mL Ampicillin (Fermentas) for 6h at 37°C. Nb expression was induced overnight with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) while shaking at 200 rpm. Cultures were centrifuged for 10 min at 1643xg and 4°C, after which the bacterial pellet was frozen at −80°C. One day later, 2 mL phosphate buffered saline (PBS, Sigma-Aldrich) was added to the thawed bacterial pellets and pellets were stirred for 30 min at 4°C. The released periplasmic proteins were collected by centrifugation for 20 minutes at 730 × g and 4°C, followed by 0.22 μm filtration (Millipore).

These PEs were used to evaluate interaction with mouse and human PD-L1 (i) in ELISA to assess qualitative binding to immobilised recombinant PD-L1 proteins; (ii) using SPR to rank Nbs for binding to recombinant PD-L1 proteins; (iii) in flow cytometry to assess binding to HEK293T cells transfected with PD-L1 cDNAs. ELISA on PEs are performed as described elsewhere [52]. SPR and flow cytometry analyses of PEs are described below.