(169c) Novel Screening Strategies for Engineering Proteases That Cleave Therapeutic Targets

In earlier studies our lab had developed technologies for the isolation of endopeptidases that recognize selectively a particular peptide sequence, via the screening of E.coli expressed libraries by FACS [1]. However, for many applications it is essential to identify enzymes that recognize and cleave at accessible sites within a folded protein. Here we report a new and very powerful system for the screening of large libraries of mutant proteases and the isolation of variants that selectively cleave a desired protein domain or loop region. Progress towards the engineering of enodpeptidase variants that cleave TNF-alpha and may be employed to inactivate this powerful inflammatory molecule irreversibly, rather via reversible binding, will be discussed. Potentially, this method can be applied to the engineering of selective endopeptidases to desired protein domain substrates.

[1] N. Varadarajan, S. Rodriguez, B. Y. Hwang, G. Georgiou, B. L. Iverson, Nat. Chem. Biol. 2008, 4, 290