The broad, long-term objective of the proposed research is to generate novel proteins that have virtually any desired property.
The specific aims of this proposal are: To construct a bi-functional tRNA that covalently links a protein to the mRNA that encodes it during in vitro protein synthesis. To use this tRNA in an in vitro selection process to isolate a monomeric form of streptavidin that has a high but reversible affinity for biotin. Every tRNA has the ability to transiently connect a protein to its encoding mRNA. By changing one atom in yeast tRNA/Phe, a tRNA that permanently links a protein to itself is made. In the ribosome, this protein-tRNA fusion can be directed cross-linked to its encoding mRNA.. The tRNA will be used to generate a large number of streptavidin variants (10/13-10/15) that have their genetic material attached. Monomeric variants that have strong but reversible biotin binding will be isolated from this pool but in-vitro selection. Such variants will be useful for a variety of biotech applications. Examination of the proteins will also provide insights into the requirements for high affinity protein oligomerization. In addition, the approach developed in this proposal can be used by others to refine the properties of other proteins or to create new proteins for medical applications.
