Journal: bioRxiv

Article Title: In silico evolution of protein binders with deep learning models for structure prediction and sequence design

doi: 10.1101/2023.05.03.539278

Figure Lengend Snippet: In the cell assay, competitive inhibition of the native PD-1:PD-L1 interaction drives luciferase expression, which is detected by monitoring luminescence. (A) The competitive binding of various masked PD-L1 antagonists (as indicated in the color key) were measured as a function of protein concentration with (right) and without (left) protease treatment. The activity of HA-PD1 without a mask (“No”) is shown for reference in black. Two technical replicates from a single experiment, presented as mean ± SEM are shown. (B) IC50s of the masked antagonists from the cell surface assay with (squares) and without (circles) protease treatment. Additional data from two independent experiments are included in the Supporting Information (Fig. S6). (C) Association rate constants (k on ) and dissociation rate constants (k off ) for the masked antagonists binding to PD-L1 with (squares) and without (circles) protease treatment. Binding measurements were made with SPR with biotinylated PD-L1 immobilized on a NeutrAvidin chip (Fig. S7). Reported error represents SD from at least 3 independent experiments except for MA4 (n = 1), MA16 (n = 1), and MA20 (n = 2).

Article Snippet: Briefly, biotinylated PD-L1 (Sino Biological, 10084-H08H-B, reconstituted according to manufacturer’s protocol) was diluted to 500 ng/mL in PBST and immobilized onto a Series S NeutrAvidin (NA) Sensor Chip (Cytiva, 29407997) to a response (RU) of ∼100.

Techniques: Inhibition, Luciferase, Expressing, Binding Assay, Protein Concentration, Activity Assay

Journal: bioRxiv

Article Title: In silico evolution of protein binders with deep learning models for structure prediction and sequence design

doi: 10.1101/2023.05.03.539278

Figure Lengend Snippet: (A) Circular dichroism (CD) spectra and (B) temperature melts of various AiDs. (C) Single-cycle SPR sensorgrams for designed AiDs (colored line) and select mutant (grey line). Biotinylated HA-PD1 was immobilized on a NeutrAvidin chip to a level of ∼250 response units (RUs). The AiDs and mutants were injected at the indicated concentrations and the data were fit to a 1:1 binding model (black). The indicated K D values are the average from three or more binding measurements (Table S4, Fig. S10, S12).

Article Snippet: Briefly, biotinylated PD-L1 (Sino Biological, 10084-H08H-B, reconstituted according to manufacturer’s protocol) was diluted to 500 ng/mL in PBST and immobilized onto a Series S NeutrAvidin (NA) Sensor Chip (Cytiva, 29407997) to a response (RU) of ∼100.

Techniques: Circular Dichroism, Mutagenesis, Injection, Binding Assay