| Protein engineering techniques can be used to create novel allosterically regulated enzymes by fusing two naturally occurring proteins so that the activity of one protein is physically linked and dependent on the other protein. This type of protein is called a "protein switch", as the activating molecule switches the enzyme from an inactive to an active state. The goal of this research was to determine principles to aid in the design and development of protein switches beyond the original proof-of-principle prototype which was created previously in the Ostermeier lab by fusing maltose binding protein and TEM-1 beta-lactamase. A new method for creating the protein fusions has been developed and was used to facilitate the creation of novel protein switches in which a circularly permuted form of TEM-1 beta-lactamase was fused to either the P. furiosus maltose binding protein, the E. coli glucose binding protein, or the E. coli ribose binding protein. The ability to improve protein switches using standard directed evolution techniques was also demonstrated. Engineering protein switches may help elucidate some of the basic principles of allostery as well as increase our ability to harness them for applications. |
