Phage-displayed combinatorial peptide libraries in fusion to beta-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications.
Phage-displayed combinatorial peptide libraries in fusion to beta-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications.
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DOI：
10.2174/138620710790218258
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发表时间：
2010-01
影响因子：
1.8
通讯作者：
Krag DN
中科院分区：
文献类型：
--
作者：
Shukla GS;Krag DN
Phage-display selection of combinatorial libraries is a powerful technique for identifying binding ligands against desired targets. Evaluation of target binding capacity of multiple clones recovered from phage display selection to a specific target is laborious, time-consuming, and a rate-limiting step. We constructed phage-display combinatorial peptide libraries in fusion with a β-lactamase enzyme, which acts as a reporter. Linear dodecapeptide and cysteine-constrained decapeptide libraries were created at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase). The overall and positional diversity of amino acids in both libraries was similar to other phage-display systems. The libraries were selected against the extracellular domain of ErbB2 receptor (ErbB2ECD). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. We used β-lactamase enzyme activity-based assays for sample normalizations and clone binding evaluation. Clones were identified that bound to purified ErbB2ECD and ErbB2-overexpressing cell-lines. The peptide sequences of the selected binding clones shared significant motifs with several rationally designed peptide mimetics and phage-display derived peptides that have been reported to bind ErbB2ECD. β-Lactamase fusion to peptides saved time and resources otherwise required by the phage-ELISA of a typical phage display screening protocol. The β-lactamase enzyme assay protocols is a one-step process that does not require secondary proteins, several steps of lengthy incubations, or washings and can be finished in a few minutes instead of hours. The clone screening protocol can be adopted for a high throughput platform. Target-specific β-lactamase-linked affinity reagents selected by this procedure can be produced in bulk, purified, and used, without any modification, for a variety of downstream applications, including targeted prodrug therapy.
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影响因子：
2.7
作者：
Hribar, Gorazd;Smilovic, Vanja;Gaberc-Porekar, Vladka
通讯作者：
Gaberc-Porekar, Vladka
影响因子：
15
作者：
Gao, WZ;Xing, BG;Rao, JH
通讯作者：
Rao, JH
DOI：
10.1016/0167-4838(82)90287-4
发表时间：
1982-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
DUEZ, C;FRERE, JM;DIERICKX, L
通讯作者：
DIERICKX, L
影响因子：
4.8
作者：
Kay, BK;Kasanov, J;Yamabhai, M
通讯作者：
Yamabhai, M
影响因子：
17.3
作者：
Campbell, RE
通讯作者：
Campbell, RE