Enhancing the promiscuity of a member of the Caspase protease family by rational design.

: The N-terminal cleavage of fusion tags to restore the native N-terminus of recombinant proteins is a challenging task and up to today, protocols need to be optimised for different proteins individually. Within this work, we present a novel protease that was designed in-silico to yield enhanced promiscuity towards different N-terminal amino acids. Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease. These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations. The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.Both mutants showed not only enhanced activities towards branched amino acids but also smaller, unbranched amino acids. We believe that the created mutants constitute an important step towards generalised procedures to restore original N-termini of recombinant fusion proteins. This article is protected by copyright. All rights reserved.