Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.
Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.
Blaise
Lydia
L
Dyax s.a., Boulevard du Rectorat 27B, Sart Tilman, 4000 Liège 1, Belgium.
Wehnert
Anita
A
Steukers
Mieke P G
MP
van den Beucken
Twan
T
Hoogenboom
Hennie R
HR
Hufton
Simon E
SE
D000915:Antibody Affinity; D002473:Cell Wall / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004171:Diploidy; D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006238:Haploidy; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009154:Mutation; D019151:Peptide Library; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019809:Streptavidin / Q000276:immunology
D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D019809:Streptavidin