Selection of a high affinity angiogenin-binding peptide from a peptide library displayed on phage coat protein.
Although the phage-displayed peptide libraries have been widely used for the biopanning of peptide specific for various types of target molecules, the selected peptides often have affinities too low for practical purposes. In this report, selection of a high affinity peptide ligand specific for human angiogenin is described. We constructed a random dodecapeptide library displayed on the gene III protein of filamentous bacteriophage by use of a phagemid. The peptide insert was flanked by two cysteines to constrain the peptide structure by a disulfide bond. Phages were collected from 1.5 x 10(3) independent transformants. After the library phages were allowed to bind to the human angiogenin coated on a plate, the phages bound to the actin-binding site of angiogenin were selectively eluted with actin. The activity of each phage clone was estimated and three high affinity phage clones were identified. The peptides displayed by the three phage clones were synthesized as fusion proteins with Escherichia coli maltose-binding protein, and used for the quantitative estimation of their affinities. By this procedure, we were able to select a peptide having a dissociation constant of about 60 nM.
Choi
S J
SJ
Department of Chemistry, Kangnung National University, Korea.
Ahn
M
M
Lee
J S
JS
Jung
W J
WJ
D018528:ATP-Binding Cassette Transporters; D000199:Actins / Q000378:metabolism; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D019151:Peptide Library; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000199:Actins; D036022:Capsid Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; C461599:maltose transport system, E coli; D008320:Maltose; C046366:angiogenin; D012259:Ribonuclease, Pancreatic