Proteases exhibiting high catalytic activity and selectivity for a specific peptide sequence can be extremely valuable for therapeutic purposes, as a means for degrading specific molecular targets for protein profiling in proteomic applications, and for bioprocessing. However, the design of proteases that recognize and cleave only a desired sequence is a very difficult problem and has remained a significant challenge. We have employed structure based sequence randomization and directed evolution couplesd with quantitative FACS screening for the engineering of highly active proteases with “restriction enzyme-like” selectivity. Key to this work was the development of methods for the screening of very large libraries of protease mutants for high activity towards selection substrates together with counterselection for lack of cleavage of other similar sequences. In this manner we have developed a set of specific dipeptidases based on the E.coli surface enzyme OmpT. In addition, efforts to engineer mammalian proteases such as chymotrypsin and cathespin and to impart allosteric activation of the catalytic activity will be described.

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