Fig. 2: Selection of monobodies against D-MCP-1 using TRAP display.

A Progress of the TRAP display selection. After each round of selection, the recovered cDNA was quantified by real-time PCR. The recovery of cDNA (%) was calculated by dividing the amount of cDNA recovered after the pull-down with D-MCP-1 by the amount of PuL in the translation mixture. From the sixth round, the selection pressure was increased by decreasing the target concentration from 25 nM to 2 nM. B Determination of kinetic parameters of Mb5 and Mb8 by BLI. D-MCP-1 was immobilized on a streptavidin-sensor chip, and Nus-Tag fused Mb5 or Mb8 (0.062, 0.125, 0.25, 0.50, 1.0 µM) were used in the kinetic analysis. The data were fitted to a 1:1 binding model. C Sequences and spatial arrangement of saturation mutagenesis libraries for affinity maturation of Mb5. Saturation mutagenesis (X) was introduced using NNK codons (N = A, C, G, T; K = G or T; 32 codons/20 aa) at six consecutive residues in the BC and FG loops. D Progress of TRAP display selection at each library in the affinity maturation. In the fifth round selection, selection stringency was increased by extending washing time was applied against mixed library derived from libraries A, B, and C. E The probability of amino acids at each position in the loops of the selected clones shown by WebLogo. F Determination of kinetic parameters of three Mb5 mutants by BLI. D-MCP-1 was immobilized on a streptavidin-sensor chip, and Mb5-9, 5-11, 5-12 (2.5, 5.0, 10, 20, 40 nM) were used in the kinetic analysis. The data are fitted to a 1:1 binding model. Abbreviations: BLI, Bio-layer interferometry; Lib, library; TRAP, transcription–translation coupled with association of puromycin linker. Source data are provided as a Source Data file.