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Directed evolution comprises two distinct steps that are typically
applied in an iterative fashion: (1) generating molecular diversity
and (2) finding among the ensemble of mutant sequences those
proteins that perform the desired fu- tion according to the
specified criteria. In many ways, the second step is the most
challenging. No matter how cleverly designed or diverse the
starting library, without an effective screening strategy the
ability to isolate useful clones is severely diminished. The best
screens are (1) high throughput, to increase the likelihood that
useful clones will be found; (2) sufficiently sen- tive (i. e. ,
good signal to noise) to allow the isolation of lower activity
clones early in evolution; (3) sufficiently reproducible to allow
one to find small improvements; (4) robust, which means that the
signal afforded by active clones is not dependent on
difficult-to-control environmental variables; and, most
importantly, (5) sensitive to the desired function. Regarding this
last point, almost anyone who has attempted a directed evolution
experiment has learned firsthand the truth of the dictum "you get
what you screen for. " The protocols in Directed Enzyme Evolution
describe a series of detailed p- cedures of proven utility for
directed evolution purposes. The volume begins with several
selection strategies for enzyme evolution and continues with assay
methods that can be used to screen enzyme libraries. Genetic
selections offer the advantage that functional proteins can be
isolated from very large libraries s- ply by growing a population
of cells under selective conditions.
Biological systems are very special substrates for
engineering-uniquely the products of evolution, they are easily
redesigned by similar approaches. A simple algorithm of iterative
cycles of diversification and selection, evolution works at all
scales, from single molecules to whole ecosystems. In the little
more than a decade since the first reported applications of
evolutionary design to enzyme engineering, directed evolution has
matured to the point where it now represents the centerpiece of
industrial biocatalyst development and is being practiced by
thousands of academic and industrial scientists in com- nies and
universities around the world. The appeal of directed evolution is
easy to understand: it is conceptually straightforward, it can be
practiced without any special instrumentation and, most important,
it frequently yields useful solutions, many of which are totally
unanticipated. Directed evolution has r- dered protein engineering
readily accessible to a broad audience of scientists and engineers
who wish to tailor a myriad of protein properties, including th-
mal and solvent stability, enzyme selectivity, specific activity,
protease s- ceptibility, allosteric control of protein function,
ligand binding, transcriptional activation, and solubility.
Furthermore, the range of applications has expanded to the
engineering of more complex functions such as those performed by m-
tiple proteins acting in concert (in biosynthetic pathways) or as
part of mac- molecular complexes and biological networks.
Biological systems are very special substrates for
engineering-uniquely the products of evolution, they are easily
redesigned by similar approaches. A simple algorithm of iterative
cycles of diversification and selection, evolution works at all
scales, from single molecules to whole ecosystems. In the little
more than a decade since the first reported applications of
evolutionary design to enzyme engineering, directed evolution has
matured to the point where it now represents the centerpiece of
industrial biocatalyst development and is being practiced by
thousands of academic and industrial scientists in com- nies and
universities around the world. The appeal of directed evolution is
easy to understand: it is conceptually straightforward, it can be
practiced without any special instrumentation and, most important,
it frequently yields useful solutions, many of which are totally
unanticipated. Directed evolution has r- dered protein engineering
readily accessible to a broad audience of scientists and engineers
who wish to tailor a myriad of protein properties, including th-
mal and solvent stability, enzyme selectivity, specific activity,
protease s- ceptibility, allosteric control of protein function,
ligand binding, transcriptional activation, and solubility.
Furthermore, the range of applications has expanded to the
engineering of more complex functions such as those performed by m-
tiple proteins acting in concert (in biosynthetic pathways) or as
part of mac- molecular complexes and biological networks.
Directed evolution comprises two distinct steps that are typically
applied in an iterative fashion: (1) generating molecular diversity
and (2) finding among the ensemble of mutant sequences those
proteins that perform the desired fu- tion according to the
specified criteria. In many ways, the second step is the most
challenging. No matter how cleverly designed or diverse the
starting library, without an effective screening strategy the
ability to isolate useful clones is severely diminished. The best
screens are (1) high throughput, to increase the likelihood that
useful clones will be found; (2) sufficiently sen- tive (i. e. ,
good signal to noise) to allow the isolation of lower activity
clones early in evolution; (3) sufficiently reproducible to allow
one to find small improvements; (4) robust, which means that the
signal afforded by active clones is not dependent on
difficult-to-control environmental variables; and, most
importantly, (5) sensitive to the desired function. Regarding this
last point, almost anyone who has attempted a directed evolution
experiment has learned firsthand the truth of the dictum "you get
what you screen for. " The protocols in Directed Enzyme Evolution
describe a series of detailed p- cedures of proven utility for
directed evolution purposes. The volume begins with several
selection strategies for enzyme evolution and continues with assay
methods that can be used to screen enzyme libraries. Genetic
selections offer the advantage that functional proteins can be
isolated from very large libraries s- ply by growing a population
of cells under selective conditions.
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