Directed Evolution Library Creation: Methods and Protocols

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 protein evolution-a powerful technique for the discovery of new enzymes and therapeutic proteins-has matured to the point that it is now the centerpiece of enzyme engineering, where it has catalyzed the development of numerous experimental methods. In Directed Evolution Library Creation: Methods and Protocols, seasoned practitioners from many leading laboratories share their best experimental protocols for the generation of molecular diversity. Described in step-by-step detail to ensure experimental success, these protocols include readily reproducible methods for random mutagenesis of entire genes or segments of genes, for homologous and nonhomologous recombination, and for constructing in vivo libraries in bacteria and yeast. In addition to the various protocols for creating libraries, this volume also describes ways to analyze libraries, particularly those made by recombination. An accompanying volume, Directed Enzyme Evolution: Screening and Selection Methods (ISBN: 1-58829-286-X), is devoted entirely to selection and screening methods that can be applied to the directed evolution of enzymes.
Taken together, Directed Evolution Library Creation: Methods and Protocols and Directed Enzyme Evolution: Screening and Selection Methods capture for newcomers and more experienced investigators alike all the key methods for using directed protein evolution to better understand protein structure-function relationships, to discover new enzymes and therapeutic proteins, and to design new assays suitable for specific applications.