A METHOD FOR DIRECTED EVOLUTION AND FUNCTIONAL CLONING OF ENZYMES

A general scheme is described for the in vitro evolution of protein ca talysts in a biologically amplifiable system, Substrate is covalently and site specifically attached by a flexible tether to the pIII coat p rotein of a filamentous phage that also displays the catalyst. Intramo lecular conversion of substrate to product provides a basis for select ing active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of fa ctors influencing the selection efficiency is presented, and it is sho wn that phage displaying staphylococcal nuclease can be enriched 100-f old in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyz e specific reactions (e.g., glycosyl transfer, sequence-specific prote olysis or phosphorylation, polymerization, etc.) rather than their seq uence- or structural homology to known enzymes.