Figure 1. Engineering of mouse SCF partial agonist.

(A) Schematic strategy to decouple SCF efficacy and toxicity. Crystal structure of SCF/c-Kit complex was obtained from PDB: 2E9W. (B) Overlay of size exclusion chromatograms. SCF and SCF F63A (SCFa) elution volumes were used to approximate their respective molecular weights based on a standard curve. (C) On-yeast competitive blocking of mouse SCF/c-Kit interaction by soluble wild-type SCF or SCFa. Yeast expressing wild-type SCF were stained with 20 nM fluorescently-labeled mKitD1-3 tetramers in the presence of unlabeled soluble SCF variants. Data represent the mean ± SEM in duplicates and are representative of two independent experiments. (D) Histogram overlays assessing mKit staining of the “second-generation” SCF library. The post-selection libraries were stained with 100 nM labeled mKitD1-3 then competed with 1 μM unlabeled mKitD1-3 for 60 min. (B and C) MFI = mean fluorescence intensity. (E) Summary of amino acid sequences of engineered high-affinity SCF variants. The position of mutated residues and their corresponding sequences in wild-type SCF are denoted at the top of the table. Red text color indicates the consensus mutations. (F) Representative SPR sensorgrams of the indicated SCF variants binding to immobilized mKitD1-3. See also Figure S1 and S2.