Abstract
Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.
Highlights
The number of combinations of mutations that can theoretically be made in a protein of 100 amino acids works out to more than 10130 possible combinations
URMAC (UnRestricted Mutagenesis and Cloning)[106] offers several advantages: it is well suited for large plasmids, having been validated on targets up to 17 kb, and it can be used for deletions, insertions and substitutions
(i) TRIAD taps into solutions that might not be accessible using libraries of point mutants through the efficient random incorporation of InDels (ii) offers the possibility to create libraries focused on a specific region of a protein, (iii) compared to other insertion and deletion methods, it solves the problem of frame shifting, (iv) it does not require the use of highly specialized equipment
Summary
The number of combinations of mutations that can theoretically be made in a protein of 100 amino acids works out to more than 10130 possible combinations. The most popular application of rational recombination is SCHEMA, which involves the guided recombination of fragments of homologous genes from the same family, giving rise to chimeric proteins.[19] SCHEMA is of interest because it is characterized by somewhat smaller theoretical library sizes (∼103–104 variants) than other recombination methods[20,21,22,23] while generating diverse libraries with a higher proportion of functional variants than randomization methods.[8,24] While these options are powerful and widely adopted, our review emphasizes focused approaches.
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