An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries

Abstract

Enzymes have become an integral part of modern bioscience, however, in their natural state, their utility is often limited. Great strides have been made in developing methods to rationally modify proteins through structural and mechanistic data, directed evolution is still the most successful means of engineering proteins with catalytic efficiency approaching those of natural enzymes. However, the application of directed evolution is often limited by the ability to develop a robust, high-throughput means of screening mutagenic libraries for enhanced function.;In chapter 2, we developed a novel in vitro compartmentalization-based bead-display method for the screening of bond-forming enzymes. We have demonstrated that this method is easily adapted for new enzymes, and that it is amenable to screening libraries of up to 1012 variants. To validate our method, in only 5 rounds we enriched a library of 1010 variants of BirA for the ability to ligate desthiobiotin to the biotin acceptor peptide (BAP). In chapter 3 we use our new selection technique to engineer variants of sortase A with enhanced intracellular activity. Sortase A, an enzyme that has been demonstrated to efficiently and specifically tag proteins with small molecule fluorophores on cell surfaces, has been shown to be inactive in the intracellular environment. With a starting library of 1011, we were able to isolate a variant of sortase A that shows a 114-fold increase in catalytic efficiency in the absence of calcium ions and increased resistance to inhibition by cell lysates. We then demonstrate the ability of this variant to perform intramolecular ligations in mammalian cells.;In addition to its potential use as a reagent to label living cells, the small tag size incorporated by sortase labeling is a desirable characteristic for other applications as well. Super-resolution microscopy techniques are being developed and refined rapidly. However, there is still need for methods to tag proteins with small molecules in a manner that is both specific and efficient. In chapter 4 we demonstrate the ability of a sortase A variant to tag proteins in fixed, permeabilized cells and discuss the potential of this technique to be used in conjunction with super-resolution micrsocopy.