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. 2020 Dec 2;206(1):225–236. doi: 10.4049/jimmunol.2001004

FIGURE 2.

FIGURE 2.

Affinity maturation of ΔB7-H6 by yeast surface display. (A) Cocrystal structure of ΔB7-H6 (blue) with NKp30 extracellular domain (green). Residues of ΔB7-H6 at the interface with NKp30 and considered for focused randomization are colored in red. This illustration is based on pdb entry 4ZSO and was generated using PyMOL v0.99. (B) Designated and observed amino acid distribution for specified positions of ΔB7-H6 as determined by sequencing of 96 clones. X (ambiguous). (C) FACS selection for the enrichment of affinity-improved variants of ΔB7-H6 by yeast surface display. A two-dimensional staining strategy for correctly displayed library candidates simultaneously binding to NKp30 was employed. 1 μM, 100 nM, and 50 nM NKp30 were used for sorting rounds one, two, and three, respectively, to enrich high-affinity binders.