Directed evolution for the development of conformation-specific affinity reagents using yeast display

Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca2+ binding. In an attempt to generate conformation-pecific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca2+-bound (Ca2+-CaM) or Ca2+-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K-d = 0.8 nM) and specificity (>1000-fold) for Ca2+-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K-d = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.