Fine affinity discrimination by yeast surface display and flow cytometry.
Yeast surface display is a eucaryotic system for the directed evolution of protein binding and stability. For antibody affinity maturation, achievable single-pass enrichment factors are a critical variable. Both reliable recovery of rare clones (yield) and effective differentiation between clones of only slightly improved affinity (purity) are paramount. To validate yeast display's purification potential, trial sorting experiments were performed. The D1.3 (anti-hen egg lysozyme) single chain variable fragment antibody and a 2-fold higher affinity mutant (M3) were each displayed on the surface of Saccharomyces cerevisiae. M3-displaying cells were mixed into the D1.3-displaying cells at a ratio of 1:1000. Cells were fluorescently labeled according to antigen equilibrium binding and then sorted using a flow cytometer. Single-pass enrichment of M3-displaying cells was 125-fold (+/- 65-fold). This level of performance is achievable because of the precision and reproducibility of optimal labeling conditions. This work further demonstrates the capability of yeast display for very fine discrimination between mutant clones of similar affinity. Because large improvements in affinity typically result from combinations of small changes, this capability to identify subtle improvements is essential for rapid affinity maturation.
VanAntwerp
J J
JJ
Department of Chemical Engineering, University of Illinois, 600 South Mathews, Urbana, Illinois 61801, USA.
Wittrup
K D
KD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D000915:Antibody Affinity; D001709:Biotechnology; D002645:Chickens; D019020:Directed Molecular Evolution; D004355:Drug Stability; D005434:Flow Cytometry; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D009113:Muramidase / Q000276:immunology; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D009113:Muramidase