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for the identification of mimotopes 92 and the optimization of anti-
genic properties for large antigens. 103 A similar concept of antigen
presentation was used for the development of cell and ribosome dis-
play techniques. 104 Another popular technology, based on the use of
libraries of synthetic peptides with random sequences, 105,106 is applica-
ble only to identification of short peptide ligands and mimotopes.
The DE techniques mentioned above use heterogeneity on the
level of individual amino acid positions. This feature makes these
techniques both comprehensive and limited in application. A tech-
nique known as DNA shuffling takes advantage of recombination —
another source of heterogeneity in nature — for the generation of
protein heterogeneity, which can be used for the selection of recom-
binants with improved immunological properties. 107,108 This tech-
nique achieves rapid evolution of genes under in vitro selection pressure
through an iterative process of recombination and selection and has
been used to develop new vaccine candidates against pathogenic
agents including dengue virus, Venezuelan equine encephalitis virus,
HIV, and HBV. 109
Despite its advantages, directed evolution is rather limited in its
applications. The approach does not build QSAR knowledge and
lacks rational design. Because the knowledge base does not grow,
building a new protein requires repeating the entire DE process from
the beginning. Although the structural approach to protein engineer-
ing also makes no direct contributions to QSAR knowledge, it chal-
lenges existing concepts and identifies research gaps. The DE
approach, on the other hand, is used as a “magic wand” that may or
may not bring desired results. Because the approach does not always
yield proteins with required specifications, it has limited applicability
for building new proteins. Using the DE strategy, new protein struc-
tures can be obtained only for rather simple properties such as pro-
tein-ligand or protein-protein interactions that can be easily modeled
with very short peptides. More complex properties, such as enzymatic
activities, require scanning a much greater protein sequence space
that cannot be accessed using random sequence peptide libraries of a
manageable size. Thus, directed evolution is not protein engineering
per se , but is more aptly characterized as molecular breeding.
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