Identifying Substrates for Endothelium-Specific Tie-2 Receptor Tyrosine Kinase from Phage-Displayed Peptide Libraries for High Throughput Screening

Authors: Deng S-j.; Liu W.; Simmons C.A.; Moore J.T.; Tian G.

Source: Combinatorial Chemistry & High Throughput Screening, Volume 4, Number 6, September 2001 , pp. 525-533(9)

Publisher: Bentham Science Publishers

Abstract:

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a kcat / Km of 5.9x104 M-1 s-1. Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.

Keywords: endothelium specific tie receptor tyrosine kinase; phage displayed peptide libraries; peptide substrate specificity; dissociation enhanced lanthanide fluoroimmunoassay; radioactive plate binding rpb; time resolved fluorescent resonance energy transfe; phosphorylation; tie kinase; gultathione s transferase; polymerase chain reaction; radioactive plate binding

Language: English

Document Type: Review article

DOI: http://dx.doi.org/10.2174/088800199276958

Publication date: 2001-09-01

• Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.

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• In this Subject: Pharmacology
• By this author: Deng S-j. ;  Liu W. ;  Simmons C.A. ;  Moore J.T. ;  Tian G.

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