Directed evolution for the development of conformation-specific affinity reagents using yeast display.
Protein Eng Des Sel
; 18(11): 527-36, 2005 Nov.
Article
em En
| MEDLINE
| ID: mdl-16186140
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Conformação Proteica
/
Saccharomyces cerevisiae
/
Evolução Molecular Direcionada
/
Afinidade de Anticorpos
Limite:
Humans
Idioma:
En
Revista:
Protein Eng Des Sel
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos