Directed Evolution for the Development of Conformation-Specific Affinity Reagents Using Yeast Display

Journal Article · Mon Sep 26 04:00:00 EDT 2005 · Protein engineering, design & selection

DOI:https://doi.org/10.1093/protein/gzi060· OSTI ID:876892

Feldhaus, Jane M; Miller, Keith D; Feldhaus, Michael J; Siegel, Robert W

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.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 876892
• Report Number(s): PNNL-SA-43934; KP1102010
• Journal Information: Protein engineering, design & selection, Journal Name: Protein engineering, design & selection Journal Issue: 11 Vol. 18
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
AFFINITY
ANTIBODIES
CALMODULIN
PROTEINS
SPECIFICITY
STABILITY
YEASTS