Biological macromolecules such as proteins and DNA are essential for every life form on earth. Studies of these molecules are dependent on our ability to selectively capture them from complex biological mixtures. Antibodies have been the most widely used for selective protein and peptide capture with applications for industrial protein purification, basic biomedical research and in clinical diagnostics. Antibodies however exhibit characteristics that limit their applications. This proposal is involved with developing robust synthetic polymers for selective capture of peptides and proteins. These substances have important applications for separations, for use in biosensors, neutralization of toxins and for the development of biomedical diagnostics. The non- biological approach of molecular imprinting is used to create specific recognition sites in robust network polymers. Protein and peptide recognition is achieved by identifying an exposed domain (epitope) of the target protein, a unique nine amino acid sequence. The peptide epitope is used as the imprint molecule. We are developing two general methods for preparing imprinted polymers for protein and peptide capture, imprinted polymer films and nanosize imprinted polymer particles. Imprinted films are prepared by covalently attaching the peptide epitope to a glass or silicon surface. Monomers are then polymerized on these surfaces to produce a molecularly imprinted polymer film (MIP). Following separation from the functionalized surface, the polymer film is evaluated for its ability to capture the target protein from protein mixtures. Two methods for the preparation of MIP nanoparticles are being developed, precipitation polymerization and suspension polymerization. In these systems, epitopes are introduced with monomers in the polymerization reaction. Following isolation and dialysis, the nanoparticles are evaluated for protein and peptide affinity and specificity. In both polymer formats, films and nanoparticles, the capture is achieved under native conditions. Molecular imprinting is one of the few general, non-biological methods for creating molecular receptors. The choice of short epitopes focuses on developing capture agents for the primary structure of the peptide rather than the more complex secondary and tertiary structure of a target protein and is similar to the use of peptide fragments to generate epitope selective antibodies and synthetic materials. In addition, the capture conditions were designed to be compatible with the native protein structure. It utilizes the sequences of exposed epitopes based on known or predicted protein structure. This method requires only the peptide sequence of a small portion of the target protein for the template molecule, it does not use or need whole protein. As such, this approach provides opportunities for the capture of target proteins based only on genomic information.
Antibodies are important reagents that are used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. Antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the target of study. We propose to develop a method for producing robust, inexpensive, non-biological polymer antibodies that can be used as substitutes for native antibodies.
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