SOLID-PHASE SYNTHESIS AND CHARACTERIZATION OF CARCINOEMBRYONIC ANTIGEN (CEA) DOMAINS

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycopro tein expressed on tumors of the colon, breast, ovary, and lung, has se ven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), most of which are recognized by distinct monoclonal antibodies. To study th e individual domains, we have prepared several of the domains (N, A3, B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were pe rformed by the Fmoc method using single couplings, elevated temperatur es for both the coupling and deblocking reactions, and a flexible solv ent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature contro l and flexible solvent control during the course of the coupling react ions. Due to the large size of the peptides (84-184 residues), it was anticipated that the overall purity of the final product would not exc eed 60% even for an average coupling yield of 99.5%. Therefore, severa l of the peptides were synthesized with a His(6) ''tail'' at the amino terminus, allowing for purification on a NI-NTA chelate column. For t he most part, the purified peptides exhibited single sharp peaks by RP -HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matr ix-assisted laser desorption ionization time of flight mass spectromet ry, gave the expected amino acid analyses, N-terminal sequences, and t ryptic maps, and bound their appropriate monoclonal antibodies. The N- domain was extremely hydrophobic, requiring 6M guanidinium hydrochlori de for solubilization, the A3 domain was soluble in dilute acid, and t he B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) ar e reported, along with characterization of the 178 amino acid hive-dom ain peptide, A3-B3. Although there is no evidence for proper folding o f these domains by NMR, their ability to bind monoclonal antibodies wi th high affinity suggests that this is a plausible approach for produc ing individual domains of CEA.