Abstract
The engineering of novel and precise sequence specificity into proteases will provide an important route to the development of exciting new tools for analytical, biotechnological, and therapeutic applications. Significant progress has been made in reprogramming protease specificity, largely because of the development of high-throughput assay technologies allowing the isolation of protease variants from large libraries. For example, using directed evolution as well as other approaches, proteases have been reprogrammed to cleave substrates containing a variety of amino acids in the P1 and P1' positions including a post-translationally modified tyrosine, a specificity not yet identified in any naturally occurring protease. Together, these recent advances represent substantial progress that could soon enable the widespread application of engineered proteases.