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1.
Biotechnol Prog ; 18(2): 182-92, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11934284

RESUMO

Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.


Assuntos
Bacteriófago M13/metabolismo , Cromatografia de Afinidade/métodos , Peptídeos/metabolismo , Albumina Sérica/isolamento & purificação , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Bacteriófago M13/genética , Ensaio de Imunoadsorção Enzimática , Polarização de Fluorescência/métodos , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Peptídeos/genética , Ligação Proteica , Sensibilidade e Especificidade , Albumina Sérica/genética , Especificidade da Espécie
2.
J Biol Chem ; 278(18): 15532-40, 2003 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-12606557

RESUMO

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homolog of ACE, is a novel metallocarboxypeptidase with specificity, tissue distribution, and function distinct from those of ACE. ACE2 may play a unique role in the renin-angiotensin system and mediate cardiovascular and renal function. Here we report the discovery of ACE2 peptide inhibitors through selection of constrained peptide libraries displayed on phage. Six constrained peptide libraries were constructed and selected against FLAG-tagged ACE2 target. ACE2 peptide binders were identified and classified into five groups, based on their effects on ACE2 activity. Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants (K(i) values) from 0.38 to 1.7 microm. Peptides from the fifth class exhibited very strong inhibition, with K(i) values < 0.14 microm. The most potent inhibitor, DX600, had a K(i) of 2.8 nm. Steady-state enzyme kinetic analysis showed that these potent ACE2 inhibitors exhibited a mixed competitive and non-competitive type of inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not inhibit ACE activity, and thus were specific to ACE2. Finally, they also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivo function, thus contributing to our better understanding of the biology of cardiovascular regulation. Our results also demonstrate that library selection by phage display technology can be a rapid and efficient way to discover potent and specific protease inhibitors.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Carboxipeptidases/antagonistas & inibidores , Peptídeos/farmacologia , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptidil Dipeptidase A
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