Alanine scanning and design of the secondary library.

(A) Ten residues in the BC and FG loops of the wild-type (WT) TA8 clone were individually substituted with the alanine residue, and the binding of each variant to the Lyn SH3 domain was assessed by ELBA. Observed binding values were normalized to that of WT TA8 clone, with error bars corresponding to the standard deviations of triplicate measurements. BSA served as the negative control. (B) Six positions in the TA8 were randomized with NNK codons for constructing the secondary library. The underlined residues of each loop are the positions that were diversified in the primary library, from which the TA8 was isolated.