Recombination of Compatible Substitutions by 2GenReP and InSiReP

The CompassR rule enables to identify the beneficial substitutions, which can be recombined in directed evolution with gradually improving the enzymatic properties. However, the question of how to efficiently explore the protein sequence space when ten or more beneficial substitutions are identified has not yet been addressed. Two recombination strategies 2GenReP and InSiReP employing CompassR are systematically investigated to minimize experimental efforts and maximize possible improvements. Here we describe the details of the 2GenReP and InSiReP procedure with an example of recombining 15 substitutions and discuss some important practical issues that should be considered for the application of 2GenReP and InSiReP, such as placing the substitutions into subsets. The core part of the protocol (Step1 to Step5) is transferable to other enzymes and any recombination of potential substitutions.