title: Directed evolution for the development of conformation-specific affinity reagents using yeast display.
abstract: 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 Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-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 Ca(2+)-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.
authors.affiliation: Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Weaver-Feldhaus
authors.forename: Keith D
authors.initials: KD
authors.lastname: Miller
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Feldhaus
authors.forename: Robert W
authors.initials: RW
authors.lastname: Siegel
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D009418:S100 Proteins / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D019209:Troponin C / Q000276:immunology / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D007135:Immunoglobulin Variable Region; D020033:Protein Isoforms; D009418:S100 Proteins; D019209:Troponin C