PNNL

Abstract

Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. In this paper, we present novel techniques for screening a non-immune scFv library to discover conformational-specific affinity reagents. A selection against mammalian calmodulin (CaM), a highly conserved signaling protein which undergoes structural changes upon Ca2+ binding, was undertaken. Screening strategies were used to easily isolate scFv recognizing CaM in the Ca2+-bound (Ca2+-CaM) and apo states (apo-CaM) are presented. One clone having very high affinity (Kd = 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 (Kd = 1 nM) and specificity (>300x) for apo-CaM from a parental clone with both a modest affinity and preference for that particular isoform. These results demonstrate that conformational specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.