- 2:00 PM
313e

Altering Effector Specificity in An Engineered Protein Switch by a Combination of Computational Design and Directed Evolution

Richard A. Heins1, Jin Ryoun Kim2, Loren L. Looger3, Takayuki Soka1, and Marc Ostermeier1. (1) Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, (2) Chemical and Biological Engineering, Polytechnic University, Brooklyn, NY 11201, (3) Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147

We have previously engineered a family of enzyme switches by the in-vitro recombination of two non-homologous genes. These switches were created using the genes encoding TEM1 beta-lactamase (BLA) and maltose binding protein (MBP) and exhibited maltose-dependent beta-lactamase activity. Another approach to creating protein switches is to modify existing switches so that they respond to new effectors. Increasing the affinity for the target ligand while simultaneously decreasing the affinity for the original ligand has proven difficult. We have attempted to convert our maltose-activated switch into one that is activated by sucrose. Our best sucrose-activated switch to date was derived from a cassette mutagenesis library in which five residues in the maltose-binding pocket were varied to all possible amino acids. This switch had a >15,000-fold increase in affinity for sucrose but only a 70-fold reduction in maltose affinity. We have created a second, computationally designed library in which 11 MBP residues are varied and subjected this library to a two-tiered genetic selection designed to identify switches that are specifically activated by sucrose. A comparison of the successes of both libraries will be presented.