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(Received for publication, February 22, 1996, and in revised form, March 26, 1996)
From Sandoz Pharma Ltd., Preclinical Research, CH-4002 Basel,
Switzerland
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 × 105
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.
Volume 271, Number 28,
Issue of July 12, 1996
pp. 16500-16505
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
SCREENING OF PEPTIDE LIBRARIES BY FLUORESCENCE-ACTIVATED BEAD
SORTING
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