Evolutionary Methods in Biotechnology: Clever Tricks for Directed Evolution - Hardcover

Hardcover

ISBN 10: 3527307990 ISBN 13: 9783527307999

Publisher: Wiley-VCH, 2004

Synopsis

Miniturization and high throughput assay technology have brought the power of molecular evolution to the bioscience laboratory. Applied wisely, the evolutionary approach can quickly yield the desired result even where other methods have failed.
From library generation by random or directed mutagenesis to screening and selection techniques -- the crucial steps for successful evolutionary biotechnology are described in detail in this practical guide that also includes valuable troubleshooting hints on frequently encountered problems.
Modern methods for the surface display of peptides and proteins, selective enrichment of nucleic acid aptamers and high-throughput screening of industrial biocatalysts are explained, and computer-based methods for in silico protein and RNA engineering are described as an alternative to in vitro approaches. A special section covers the patenting regulations with regard to biotechnological innovations derived from directed evolution.
As an added bonus, a CD-ROM is included that contains software tools for library design, selection of mutagenesis positions, and various predictive algorithms.
In short, this practice oriented handbook is an indispensable tool for every scientist working in this interdisciplinary research area.

About this title

About the Author

Susanne Brakmann is head of the junior research group "Applied Molecular Evolution" at the University of Leipzig (Germany) and a Member of the Biotechnological-Biomedical Center of Leipzig. She studied Chemistry at the Technical University of Braunschweig where she received her diploma in 1988, moving afterwards to the University of Karlsruhe to work on her thesis under the supervision of Reinhold Tacke (Ph. D. 1991). She was postdoctoral fellow at the Max-Planck-Institute for Biophysical Chemistry in Gottingen where she worked with Manfred Eigen before she moved to Leipzig in 2001. She is interested in directed evolution as a tool for understanding and optimizing enzyme functions, focusing on nucleic acid polymerases and their biotechnological applications.

Andreas Schwienhorst is a group leader at the Institute of Microbiology and Genetics at the University of Gottingen (Germany). He was born in Warendorf and did his studies of Biochemistry and Prehistory in Munster and Gottingen. His thesis was carried out at the Max-Planck-Institute for Biophysical Chemistry under the supervision of Manfred Eigen. He was a visiting scientist at the Salk Institute, La Jolla and the Los Alamos National Laboratory (USA), then took a position as a group leader at the Institute for Molecular Biotechnology before moving to the University of Gottingen in 1997. In 1998 he received the Biofuture Award. Currently, he is interested in the discovery of novel targets for drug intervention as well as in methods of molecular evolution and their applications in drug discovery and catalysis.

Excerpt. © Reprinted by permission. All rights reserved.

Evolutionary Methods in Biotechnology

John Wiley & Sons

Copyright © 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All right reserved.
ISBN: 978-3-527-30799-9

Chapter One

Introduction

Susanne Brakmann and Andreas Schwienhorst

Since the landmark papers of Manfred Eigen and Sol Spiegelman, the concept of Darwinian evolution has had a major impact on the design of biomolecules with tailored properties. 'Directed evolution', 'applied evolution', and 'evolutionary biotechnology' are different expressions that all describe an 'evolutionary' type of optimization strategy that comprises several cycles each consisting of (1) molecular library preparation to create the desired molecular diversity, (2) functional selection or screening, and (3) error-prone amplification or chemical modification of selected species to generate a new library of molecules (Fig.1.1). The ultimate goal is to identify molecular species that are well-adapted to a given profile of defined demands. Biocatalysts, for example, may be generated to exhibit high processivity, enantioselectivity, or tolerance to high temperatures or organic solvents.

The book presented here is intended as a practical state-of-the-art compilation of methods related to the topic of directed evolution and hence is complementary to the recent successful book Directed Molecular Evolution of Proteins. The methods are described in sufficient detail to serve as 'recipes' in a 'cookbook'. They are easy to follow by laboratory staff, from the technical assistant to the postdoctoral academic or industrial specialist.

The sequence of chapters mirrors the steps in a standard directed-evolution experiment. In the beginning, various methods for the creation of molecular diversity are considered. S. Brakmann and B.F. Lindemann (Chapter 2) present protocols for the generation of mutant libraries by random mutagenesis. Two chapters deal with the particularly powerful approach of in-vitro recombination. H. Suenaga, M. Goto, and K. Furukawa (Chapter 3) describe the application of DNA shuffling, and M. Ninkovic (Chapter 4) presents DNA recombination by the StEP method.

Next, several chapters are concerned with techniques of selection and/or mass screening technologies. T. Adams, H.-U. Schmoldt, and H. Kolmar (Chapter 5) describe the FACS-based screening of combinatorial peptide and protein libraries. P. Soumillion (Chapter 6) presents some of the latest developments in the selection of phage-displayed enzymes. In Chapter 7, H. Fickert, H. Betat, and U. Hahn provide methods for the selection of specific target-binding nucleic acids, i. e., aptamers. Related methods for the generation of catalytic nucleic acids are described by B.L. Holley and B.E. Eaton (Chapter 8). The part on functional selection and screening closes with a description of high-throughput screening approaches, in particular, to produce enantioselective industrial biocatalysts, provided by M.T. Reetz (Chapter 9).

Combinatorial mutagenesis easily produces a degree of molecular diversity that far exceeds the number of different proteins or functional nucleic acids that can be produced in a single experiment. As the number n of randomized amino acid positions in a protein grows, the number of possible combinations increases as 20ⁿ. Hence, complete coverage of a library with 9 randomized positions requires a library size well above 10¹¹ molecules. Since in a standard random library, functional molecules are usually highly diluted in a large background of nonfunctional, e. g., misfolded, molecules, it may be meaningful to restrict variations to a certain subset of promising molecules. Three chapters deal with theoretical computer-based methods to predict these promising molecular species. D. Tomandl and A. Schwienhorst (Chapter 10) report a 'doping' algorithm that helps to design random codons for only subsets of amino acids, at the same time minimizing stop codons. M. Wiederstein, P. Lackner, F. Kienberger, and M.J. Sippl (Chapter 11) provide algorithms to predict (mutant) protein structures as a means of in silico mutagenesis, e. g., to enhance the probability of generating properly folded mutant proteins. C. Flamm, I.L. Hofacker, and P.F. Stadler (Chapter 12) pursue a similar goal concerning functional nucleic acids and provide various in silico tools to predict RNA folding.

In the past 10 years, directed evolution has gained considerable attention as a commercially important strategy for rapidly designing molecules with properties tailored for the biotechnological and pharmaceutical market. Therefore, legal protection of methods and molecules has become an important issue. Hence, the book closes with Chapter 13, by M. Leimkhler and H.W. Meyers on patenting issues in evolutionary biotechnology.

Since the first evolution experiments by Sol Spiegelman, Manfred Eigen, and coworkers, the field of directed evolution itself has evolved into a plethora of different methodologies that can hardly be covered comprehensively in a standard textbook. We nevertheless tried to provide a collection of protocols useful to the novice as well as to the scientist experienced in the field. We hope to provide a practical starting point and at the same time inspire scientists to develop their own variations on the evolutionary theme.

We thank all the authors for their contributions, and Peter Glitz and Frank Weinreich of Wiley-VCH for their help in publishing this book.

(Continues...)

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Edition details

• Publisher: Wiley-VCH
• Publication date: 2004
• Language: English
• ISBN 10: 3527307990
• ISBN 13: 9783527307999
• Binding: Hardcover
• Edition number: 1
• Number of pages: 227
• Editor: Brakmann Susanne, Schwienhorst Andreas