A book chapter for practically applying two recombination strategies 2GenReP and InSiReP in protein engineering campaigns
Congratulations Haiyang (Ocean) Cui on his recent publication!
The CompassR rule enables to identify the beneficial substitutions, which can be recombined in directed evolution with gradually improving the enzymatic properties (see our previous publications https://doi.org/10.1002/chem.201903994, https://doi.org/10.1039/D1GC00763G). 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. In Haiyang (Ocean)´s new book chapter, 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 (Step 1 to Step 5) is transferable to other enzymes and any recombination of potential substitutions.
This work was performed in the Computational Biology division and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169). Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship.
To learn more, please access the full paper on publications and patents and
Cui, Haiyang, Mehdi D. Davari, and Ulrich Schwaneberg. "Recombination of Compatible Substitutions by 2GenReP and InSiReP." In Enzyme Engineering, pp. 71-81. Humana, New York, NY, 2022. https://doi.org/10.1007/978-1-0716-1826-4_5