Biology Reference
In-Depth Information
or
template for engineering. In other cases more than one parent serves as the
starting point for engineering chimeric proteins (i.e. proteins whose gene sequences are
hybrids of more than one gene, resulting from gene recombination). In addition to the
citations provided in context throughout this chapter, we refer readers interested in more
methods and/or details of experimental protocols in protein engineering to several volumes
dedicated to this field.
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parent
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PROTEIN ENGINEERING METHODS
As depicted in
Figure 2.1
, methods of protein engineering can be broadly categorized as rational
design and combinatorial design or directed evolution, although a combination of these is
often used. Rational design involves the use of protein sequence, structure, and function
information, and any known relationships between these, to devise specific mutagenesis
Rational design
Directed evolution
Wild-type / Parent gene
Additional parent gene(s)
or
Structure analysis/
modeling
Random
mutagenesis
Recombination
mutant 1
24
mutant 2
site-directed mutagenesis
/ gene fusion
Gene library ligation and
transformation
mutant 1
mutant 2
Ligation and
transformation
Protein library expression
High-throughput
screen / selection
Protein expression
and characterization
Evolved variant(s)
Evolved gene(s)
FIGURE 2.1
Approaches to engineer proteins can be broadly categorized as rational design or directed evolution. Rational design involves
the use of sequence/structure/function information to predict site-directed mutagenesis strategies (e.g. point mutations or
gene fusions) that will improve properties of interest. The protein
'
s gene sequence is modified accordingly, and the protein
variant is expressed and characterized. Directed evolution involves the generation of gene libraries based on one or more
parent gene sequence. The library of mutants are expressed and assayed in a high-throughput screen. Improved variants are
selected and the process can be repeated, thus mimicking natural evolution. As described in the text, there are many
variations on these methods, including semirational design and in vitro expression.
