MOTIVATION: Current techniques of protein engineering focus mostly on re-designing small targeted regions or defined structural scaffolds rather than constructing combinatorial libraries of versatile compositions and lengths. This is a missed opportunity because combinatorial libraries are emerging as a vital source of novel functional proteins and are of interest in diverse research areas. RESULTS: Here, we present a computational tool for Combinatorial Library Design (CoLiDe) offering precise control over protein sequence composition, length and diversity. The algorithm uses evolutionary approach to provide solutions to combinatorial libraries of degenerate DNA templates. We demonstrate its performance and precision using four different input alphabet distribution on different sequence lengths. In addition, a model design and experimental pipeline for protein library expression and purification is presented, providing a proof-of-concept that our protocol can be used to prepare purified protein library samples of up to 1011-1012 unique sequences. CoLiDe presents a composition-centric approach to protein design towards different functional phenomena. AVAILABILITYAND IMPLEMENTATION: CoLiDe is implemented in Python and freely available at https://github.com/voracva1/CoLiDe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Department of Biochemistry Faculty of Science Charles University 128 00 Prague 2 Czech Republic

Department of Cell Biology Faculty of Science Charles University Biocev Prague Czech Republic

Department of Cybernetics Center for Machine Perception Faculty of Electrical Engineering Czech Technical University Prague 166 27 Prague Czech Republic

Earth Life Science Institute Tokyo Institute of Technology Tokyo 1528550 Japan

Institute of Organic Chemistry and Biochemistry IOCB Research Centre and Gilead Sciences Academy of Sciences of the Czech Republic 166 10 Prague Czech Republic

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Building the SynBio community in the Czech Republic from the bottom up: You get what you give

Modern and prebiotic amino acids support distinct structural profiles in proteins