ausblenden:
Schlagwörter:
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Zusammenfassung:
Directed evolution is a powerful approach to study the molecular basis
of protein evolution and to engineer proteins for a wide range of
applications in synthetic organic chemistry and biotechnology. There are
many methods based on random or focused mutagenesis to engineer
successfully any protein trait. Focused approaches such as site-directed
and saturation mutagenesis have become methods of choice for improving
protein activity, selectivity, stability and many other traits because
the screening step can be practically handled (bottleneck in directed
evolution). Although novel mutagenesis methods based on CRISPR or
solid-phase gene synthesis can eliminate bias when creating protein
libraries, traditional PCR approaches, although imperfect, remain widely
used due to their ease and low cost. One of the most common approaches
in focused mutagenesis relies on NNK mutagenesis, however, the
primer-based 22c-trick and small-intelligent methods have emerged as key
tools for constructing less biased and unbiased libraries when all 20
canonical amino acids are needed for various reasons. In this
minireview, we assess studies employing such methods for library
creation and their areas of application. We also discuss the advantages
and disadvantages of both methods and provide a perspective for creating
smarter libraries.
