Evolutionary Methods in Biotechnology

Transcription

1 Evolutionary Methods in Biotechnology Clever Tricks for Directed Evolution Edited by Susanne Brakmann and Andreas Schwienhorst

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3 Evolutionary Methods in Biotechnology Edited by Susanne Brakmann and Andreas Schwienhorst

4 Further Titles of Interest G. Kahl The Dictionary of Gene Technology 3rd edition 2004, ISBN A.S. Bommarius, B.R. Riebel Biocatalysis Fundamentals and Applications 2004, ISBN T. Dingermann, D. Steinhilber, G. Folkers (eds.) Molecular Biology in Medicinal Chemistry 2003, ISBN S. Brakmann, K. Johnsson (eds.) Directed Molecular Evolution of Proteins 2002, ISBN V. Braun, F. Götz (eds.) Microbial Fundamentals of Biotechnology 2001, ISBN

5 Evolutionary Methods in Biotechnology Clever Tricks for Directed Evolution Edited by Susanne Brakmann and Andreas Schwienhorst

6 Dr. Susanne Brakmann Institut für Zoologie Angewandte Molekulare Evolution Universität Leipzig Liebigstr Leipzig, Dr. Andreas Schwienhorst Institut für Mikrobiologie und Genetik Universität Göttingen Grisebachstr Göttingen, aschwie1@gwdg.de This book was carefully produced. Nevertheless, authors, editors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No. applied for. British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library Die Deutsche Bibliothek CIP Cataloguing-in-Publication-Data: A catalogue record for this publication is available from Die Deutsche Bibliothek ISBN c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Printed on acid-free paper All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form by photoprinting, microfilm, or any other means nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition: EDV-Beratung Frank Herweg, Leutershausen Printing: betz-druck gmbh, Darmstadt Bookbinding: J. Schäffer GmbH & Co. KG, Grünstadt Printed in the Federal Republic of.

7 Contents 1 Introduction Susanne Brakmann and Andreas Schwienhorst... 1 References Generation of Mutant Libraries Using Random Mutagenesis Susanne Brakmann and Björn F. Lindemann Introduction Materials Materials for Random PCR Mutagenesis Materials for Mutator Strain Passage Protocols Protocol for Random PCR Mutagenesis According to Joyce Protocol for Mutator Strain Passage Troubleshooting References DNA Shuffling Hikaru Suenaga, Masatoshi Goto, and Kensuke Furukawa Introduction Materials For Preparation of Parental Genes For Random Fragmentation by DNase I For Collection of DNA Fragments in Specific Molecular Size Ranges For Reassembly of These Fragments by Primerless PCR For Amplification of Reassembled Products by Conventional PCR with Primers Protocol Preparation of Parental Genes Random Fragmentation by DNase I Collection of DNA Fragments in Specific Molecular Size Ranges Reassembly of These Fragments by Primerless PCR Amplification of Reassembled Products by Conventional PCR with Primers... 20

8 VI Contents 3.4 Troubleshooting Insufficient DNase I Fragmentation Little or No Product of Primerless PCR Little or No Product of PCR with Primers The Product of PCR with Primers is Multi-banded Amplification Examples References DNA Recombination Using StEP Milena Ninkovic Introduction Materials StEP PCR Purification of an Appropriate DNA Fragment Equipment Protocol Technical Tips Problem: Little or No PCR Product (Full-length Product) after PCR Problem: High Background Levels of DNA after PCR StEP in Directed Evolution References FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells Thorsten M. Adams, Hans-Ulrich Schmoldt, and Harald Kolmar Introduction Materials Escherichia coli Strains and Plasmids Liquid Media and Agar Plates Biological and Chemical Materials Equipment Protocols Verification of Cell Surface Exposure of the Passenger Protein Labeling of the Target Protein Library Construction Combinatorial Library Screening by FACS and MACS Troubleshooting Major Applications References Selection of Phage-displayed Enzymes Patrice Soumillion Introduction Materials... 50

9 Contents VII Buffers, Reagents and Consumables Strains and Vectors Protocols The Phage-enzyme Library Construction Selection Troubleshooting Phage Titers Are Not Reproducible Phage-enzymes Degrade with Time Phages Are Not Genetically Stable The out/in Ratio Does Not Increase with Selection Rounds Major Applications References Selection of Aptamers Heiko Fickert, Heike Betat, and Ulrich Hahn Introduction Materials Immobilization of Target Molecules PCR In vitro Transcription RNA Purification Selection of Aptamers Reverse Transcription Protocols Selection of RNA Aptamers Selection of 2 -Modified RNA Aptamers Selection of ssdna Aptamers Cloning and Sequencing Characterization of Aptamers Example: Isolation of Moenomycin A-specific Aptamers Troubleshooting Major Applications References Methods for Selecting Catalytic Nucleic Acids Benjamin L. Holley, and Bruce E. Eaton Introduction Materials and Equipment Protocols Generating the Starting Library Transcription Ligation Nucleic Acid-catalyzed Reactions Reverse Transcription

10 VIII Contents Partitioning Troubleshooting Major Applications References High-throughput Screening of Enantioselective Industrial Biocatalysts Manfred T. Reetz Introduction Materials and Equipment Assays Based on Mass Spectrometry Assays Based on NMR Spectrometry Assay Based on FTIR Spectroscopy Assays Based on UV/Visible Spectroscopy Enzyme-coupled UV/Visible-based Assay for Hydrolases Protocols Assays Based on Mass Spectrometry Assays Based on NMR Spectroscopy Assay Based on FTIR Spectroscopy Assays Based on UV/Visible Spectroscopy Enzyme-coupled UV/Visible-based Assay for Hydrolases Further Assays Troubleshooting Comments on the Kazlauskas Test Potential Problems when Performing Kinetic Resolution Conclusions References Computer-assisted Design of Doped Libraries Dirk Tomandl and Andreas Schwienhorst Introduction Materials Protocol Troubleshooting Major Applications References Directed in silico Mutagenesis Markus Wiederstein, Peter Lackner, Ferry Kienberger, Manfred J. Sippl Introduction Materials PDB Files Knowledge-based Potentials Polyprotein, Z-scores In silico Mutagenesis Summary

11 Contents IX 11.3 Protocol ProSa Setup and Interaction ProSa Objects Session 1 (mut script1.cmd) Session 2 (mut script2.cmd) Session 3 (mut script3.cmd) Session 4 (mut script4.cmd) Tips & Tricks Troubleshooting Major Applications References RNA Folding in silico Christoph Flamm, Ivo L. Hofacker, Peter F. Stadler Introduction Materials Typographical Conventions RNA Web Services Protocols Secondary Structures for Individual Sequences Consensus Structures of a Sample of Sequences Sequence Design Analysis of SELEX Experiments A Note for the Experts: Write your Own RNA Programs Troubleshooting Caveats References Patenting in Evolutionary Biotechnology Martina Leimkühler and Hans-Wilhelm Meyers Introduction The Nature of Patents What Can Be Patented The Requirement of Novelty The Requirement of Inventiveness The Requirement of Utility The Requirements of Enablement and Written Description Patent Prosecution Search Tools The First-to-invent Principle of the United States and Its Consequences on Laboratory Notebook Keeping Summary References Subject Index...211

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13 List of Contributors Thorsten M. Adams Institut für Mikrobiologie und Genetik Universität Göttingen Grisebachstr Göttingen Heike Betat Max-Planck-Institut für Evolutionäre Anthropologie Deutscher Platz Leipzig Susanne Brakmann Institut für Zoologie Angewandte Molekulare Evolution Universität Leipzig Liebigstr Leipzig Bruce E. Eaton Department of Chemistry North Carolina State University Raleigh, NC USA Heiko Fickert Universität Hamburg Institut für Biochemie und Lebensmittelchemie Martin-Luther-King Platz Hamburg Christoph Flamm Institut für Theoretische Chemie und Strukturbiologie Universität Wien Währingerstr Wien Austria Kensuke Furukawa Department of Bioscience and Biotechnology Kyushu University Fukuoka Japan Masatoshi Goto Department of Bioscience and Biotechnology Kyushu University Fukuoka Japan Ulrich Hahn Universität Hamburg Institut für Biochemie und Lebensmittelchemie Martin-Luther-King Platz Hamburg Ivo L. Hofacker Institut für Theoretische Chemie und Strukturbiologie Universität Wien Währingerstr Wien Austria

14 XII List of Contributors Benjamin L. Holley Department of Chemistry North Carolina State University Raleigh, NC USA Ferry Kienberger Center of Applied Molecular Engineering Institute of Chemistry and Biochemistry University of Salzburg Jakob Haringerstr Salzburg Austria Harald Kolmar Institut für Mikrobiologie und Genetik Universität Göttingen Grisebachstr Göttingen Peter Lackner Center of Applied Molecular Engineering Institute of Chemistry and Biochemistry University of Salzburg Jakob Haringerstr Salzburg Austria Martina Leimkühler Evotec OAI AG Schnackenburgallee Hamburg Björn Lindemann Bioagency AG Schnackenburgallee 116a Hamburg Hans-Wilhelm Meyers Deichmannhaus am Dom Bahnhofsvorplatz Köln Milena Ninkovic Max-Planck-Institut für Experimentelle Medizin Hermann-Rein-Str Göttingen Manfred T. Reetz Max-Planck-Institut für Kohlenforschung Kaiser-Wilhelm-Platz Mülheim/Ruhr Hans-Ulrich Schmoldt Institut für Mikrobiologie und Genetik Universität Göttingen Grisebachstr Göttingen Andreas Schwienhorst Institut für Mikrobiologie und Genetik Universität Göttingen Grisebachstr Göttingen Manfred J. Sippl Center of Applied Molecular Engineering Institute of Chemistry and Biochemistry University of Salzburg Jakob Haringerstr Salzburg Austria

15 List of Contributors XIII Patrice Soumillion Institut des Sciences de la Vie Université Catholique de Louvain Place Pasteur 1/1b 1348 Louvain-la-Neuve Belgium Peter F. Stadler Institut für Informatik Universität Leipzig Kreuzstrasse 7b Leipzig Hikaru Suenaga Institute for Biological Resources and Functions National Institute of Advanced Industrial Science and Technology Tsukuba, Ibaraki Japan Dirk Tomandl Graffinity Pharmaceuticals AG Im Neuenheimer Feld Heidelberg Markus Wiederstein Center of Applied Molecular Engineering Institute of Chemistry and Biochemistry University of Salzburg Jakob Haringerstr Salzburg Austria