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expression and screening of the chimeric progeny, further rounds of combinatorial
mutagenesis are often employed until no further improvements are obtained-this process is
termed directed evolution.
Directed evolution has achieved extraordinary success in several industrial enzymes,
resulting in 100- to over 10,000-fold enhancements of activity, altered substrate specificities,
and stabilities to environmental conditions such as acidity, temperature, and solvent
composition [24, 25]. Assembly PCR [26] as well as several other related combinatorial
methods known by their acronyms as StEP, SHIPREC, ITCHY, SCRATCHY, CLERY, and
RACHITT, are described in detail in the topic, Directed Evolution Library Creation: Methods
and Protocols [27].
2.5.4. Rational design . The mystery surrounding the way in which a one- dimensional
sequence of amino acids folds into an active three-dimensional (3D) enzyme has fascinated
researchers for decades. Now, the understanding that has emerged from their work,
summarized in several excellent topics [28-30], in combination with the crystallization and
structural analysis of a number of industrially important enzymes, is making the rational
mutagenesis of such enzymes possible.
Rational design typically begins with the computer-based structural representation of a
protein of interest, ideally informed by high-resolution, 3D structures of the protein or near
relatives obtained by x-ray crystallography or nuclear magnetic resonance (NMR)
spectroscopy. Structures of enzyme-substrate, enzyme-product, and enzyme-inhibitor
complexes are especially useful, because they identify the amino acids that comprise the
catalytic part of the enzyme, or the active site. However, simulation is also possible with
knowledge of only the primary (sequence) structure, albeit at lower fidelity, based on
similarity to homologous proteins and current understanding of protein folding kinetics and
thermodynamics. Extensive protein sequence and structure databases exist and are freely
available, most notably including Swiss- Prot and its supplement, TrEMBL, found online at
http://us.expasy.org/sprot/. Secondary and tertiary structure predictions may then be generated
by the following homology modeling software: SWISS-MODEL, an automated protein
modeling server at the GlaxoWellcome Experimental Research Station in Geneva,
Switzerland (free online at http://swissmodel.expasy.org/); PredictProtein, an online service
for sequence analysis and structure prediction maintained by the European Molecular Biology
Laboratory - Heidelberg (EMBL) and the Columbia University Bioinformatics Center
(CUBIC) at http://www.emblheidelberg.de/predictprotein/predictprotein.html; and Modeller,
maintained by the University of California at San Francisco and also free of charge to
academic researchers at http://salilab.org/modeller/modeller.html.
Once the protein of interest has a structural representation, the most promising sites for
modification can be predicted, often computationally. These are typically close to the active
site and accommodate or stabilize the proposed reaction mechanism. They are also in the
binding pocket for the substrate, or make important structural contributions to the enzyme. By
site- directed mutagenesis of the DNA that encodes these amino acids, in which a common
approach is to allow oligonucleotides bearing the desired mutation(s) to prime PCR
amplification reactions at the locus of interest, other residues can be substituted in their
place(s) [31, 21]. Amino acids are usually exchanged with one of the other 19 natural
residues, but it has recently become fashionable to incorporate so-called designer amino acids
into proteins as well. The mutated gene is then introduced into a suitable organism and
expressed, and the new protein product is partially or completely purified to reveal whether
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