The objective of this proposal is to develop a general method of introducing non-natural amino acids into proteins. Site specific mutagenesis currently allows replacement of any one of the twenty normal amino acids constituents of proteins with another. Despite the enormous value of this type of manipulation in the study of enzyme mechanisms, and other areas, it is severely limited because other groups cannot be introduced by this method. The use of a synthetic amber suppressor system (a UAG stop codon and de novo synthesized acylated t-RNAs) should allow the introduction of a variety of different groups at a single designated position in proteins. For example, it should be possible to introduce specific detection residues (e.g., fluorescence-labeled amino acids for enzyme metabolism studies), catalytic residues (e.g., metal chelating activity), linking residues (e.g., groups capable of forming covalent bonds after activation, for specific enzyme immobilization, enhanced thermal stability, or post-translational modification), cleaving residues (for proenzyme -> enzyme conversion), and receptor/cell-specific residues (for peptide delivery to specific cellular sites). The successful completion of this project would provide significant new tools for genetic engineering, enzymologists, biochemists, and bioorganic chemists.
| Bain, J D; Switzer, C (1992) Regioselective ligation of oligoribonucleotides using DNA splints. Nucleic Acids Res 20:4372 |
| Bain, J D; Diala, E S; Glabe, C G et al. (1991) Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs. Biochemistry 30:5411-21 |
