Abstract

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (k_cat/K_M > 10⁴ M^{- 1} s^{- 1}). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including GluArg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.

1. Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
2. Department of Chemical Engineering, University of Texas, Austin, Texas 78712, USA.
3. Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.

Correspondence to: George Georgiou^1,2 e-mail: gg@che.utexas.edu

Correspondence to: Brent L Iverson^1,3 e-mail: biverson@mail.utexas.edu

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