Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting.
J Biol Chem
; 271(28): 16500-5, 1996 Jul 12.
Article
em En
| MEDLINE
| ID: mdl-8663178
ABSTRACT
A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 x 10(5) individual peptides of nine amino acids in length (EPX6Y*X19X7X19X7X6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfopeptídeos
/
Domínios de Homologia de src
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
1996
Tipo de documento:
Article
País de afiliação:
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