Recombination of Single Beneficial Substitutions Obtained from Protein Engineering by Computer-Assisted Recombination...

A large number of beneficial substitutions can be obtained from a successful directed enzyme evolution campaign and/or (semi)rational design. It is expected that the recombination of some beneficial substitutions leads to a much higher degree of performance through synergistic effect. However, systematic recombination studies show that poorly performing variants are often obtained after recombination of three to four individual beneficial substitutions and this limits protein engineers to exploit nature's potential in generating better performing enzymes. Computer-assisted Recombination (CompassR) strategy allows the recombination of identified beneficial substitutions in an effective and efficient manner in order to generate active enzymes with improved performance. Here, we describe in detail the CompassR procedure with an example of recombining four substitutions and discuss some important practical issues that should be considered (such as the selection of protein structures, number of FoldX runs, evaluation of calculations) for application of the CompassR rule. The core part of this protocol (system setup, ΔΔG_fold calculation, and CompassR application) is transferable to other enzymes and any recombination of single beneficial substitutions.