期刊
CHEMBIOCHEM
卷 4, 期 4, 页码 272-276出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200390047
关键词
combinatorial chemistry; fluorescent probes; lanthanides; peptides
Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb3+ ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally clevable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K-D) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb3+ ions.
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