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Titolo:
SOLID-PHASE SYNTHESIS AND CHARACTERIZATION OF CARCINOEMBRYONIC ANTIGEN (CEA) DOMAINS
Autore:
KAPLAN BE; HEFTA LJ; BLAKE RC; SWIDEREK KM; SHIVELY JE;
Indirizzi:
CITY HOPE NATL MED CTR,BECKMAN RES INST,DEPT BIOL MOL,1450 E DUARTE RD DUARTE CA 91010 CITY HOPE NATL MED CTR,BECKMAN RES INST,DEPT IMMUNOL DUARTE CA 91010 XAVIER UNIV,COLL PHARM NEW ORLEANS LA 70125
Titolo Testata:
The journal of peptide research
fascicolo: 4, volume: 52, anno: 1998,
pagine: 249 - 260
SICI:
1397-002X(1998)52:4<249:SSACOC>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
TOTAL CHEMICAL SYNTHESIS; METAL CHELATE ADSORBENT; MONOCLONAL-ANTIBODIES; PEPTIDE-SYNTHESIS; MODEL SYSTEM; PROTEINS; SPECIFICITIES; PURIFICATION; EXPRESSION; RESINS;
Keywords:
CARCINOEMBRYONIC ANTIGEN (CEA); SOLID-PHASE SYNTHESIS; COUPLING REACTIONS; BIA CORE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
36
Recensione:
Indirizzi per estratti:
Citazione:
B.E. Kaplan et al., "SOLID-PHASE SYNTHESIS AND CHARACTERIZATION OF CARCINOEMBRYONIC ANTIGEN (CEA) DOMAINS", The journal of peptide research, 52(4), 1998, pp. 249-260

Abstract

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), mostof which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3,B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84-184 residues), it wasanticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His(6) ''tail'' at the amino terminus, allowing for purification on a NI-NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP-HPLC, migrated at their expected molecular weights by gel permeationchromatography, gave correct masses by electrospray ionization or matrix-assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N-terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N-domain was extremely hydrophobic, requiring 6M guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid hive-domain peptide, A3-B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.

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Documento generato il 30/11/20 alle ore 06:36:26