The overall goal of this SBIR grant proposal is to develop and commercialize our biochemical techniques to produce large quantities of bilogical proteins containing synthetic amino acids at predetermined sites. In Phase I, we established an efficient and robust method for the elaboration of such proteins. In Phase II, we will prepare a set of analogues of key side chain-functionalized amino acids and demonstrate that each of the amino acids can be incorporated efficiently at a single site in a protein of interest. We will make chloramphenicol acetyltransferase analogues using those synthetic amino acids to explore protein function. We will also develop a structurally novel amino acid that can be derivatized post-translationally after incorporation into a single, predetermined site of a protein, and will synthesize novel fluorescent reporter groups and demonstrate their incorporation into that site of the protein and their utility in defining protein conformation. In the latter phase of this program, we will produce mg quantities of each of two DHFR analogues to validate the overall approach. At the conclusion we will have reagents and information necessary to successfully market a robust and efficient means of analyzing the function(s) of specific amino acids in proteins and to produce modified proteins at levels significantly greater than is currently possible.
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