Main description:

This volume details basic and advanced protocols for both stages of protein engineering: the library design phase and the identification of improved variants by screening and selection. Chapters focus on enzyme engineering using rational and semi-rational approaches. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Protein Engineering: Methods and Protocols aims to aid scientists in the planning and performance of their experiments.

The chapter 'Functional Analysis of Membrane Proteins Produced by Cell-Free Translation' is open access under a CC BY 4.0 license via link.springer.com.

Contents:

1. Protein Engineering - Past, Present, and Future Stefan Lutz and Samantha M. Iamurri

Part I

2. (Semi)rational Protein Design

Ivan V. Korendovych

3. Computational Analysis of Protein Tunnels and Channels

Jan Brezovsky, Barbora Kozlikova, and Jiri Damborsky

4. YASARA - A Tool to Obtain Structural Guidance in Biocatalytic Investigations

Henrik Land and Maria Svedendahl Humble

5. A Computational Library Design Protocol for Rapid Improvement of Protein Stability - FRESCO

Hein J. Wijma, Maximilian J.L.J. Furst, and Dick B. Janssen

6. Directed Evolution of Proteins Based on Mutational Scanning

Carlos G. Acevedo-Rocha, Matteo Ferla, and Manfred T. Reetz

Part II

7. A Brief Guide to the High-throughput Expression of Directed Evolution Libraries

Ana Luisa Ribeiro, Mario Mencia, and Aurelio Hidalgo

8. Library Growth and Protein Expression: Optimal and Reproducible Microtiter Plate Expression of Recombinant Enzymes in E. coli using MTP Shakers

Sandy Schmidt, Mark Doerr, and Uwe T. Bornscheuer

9. Normalized Screening of Protein Engineering Libraries by Split-GFP Crude Cell Extract Quantification

Javier Santos-Aberturas, Mark Doerr, and Uwe T. Bornscheuer

10. Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

Srujan Kumar Dondapati, Doreen A Wustenhagen, and Stefan Kubick

PART III

11. Practical Considerations Regarding the Choice of the Best High-throughput Assay

Carolin Mugge and Robert Kouristb

12. High-throughput Screening Assays for Lipolytic Enzymes

Alexander Fulton, Marc R. Hayes, Ulrich Schwaneberg, Joerg Pietruszka, and Karl-Erich Jaeger

13. Continuous High-throughput Colorimetric Assays for -transaminases

Egon Heuson, Jean-Louis Petit, Franck Charmantray, Veronique de Berardinis, and Thierry Gefflaut

14. Colorimetric High-troughput Screening Assays for the Directed Evolution of Fungal Laccases

Isabel Pardo and Susana Camarero

15. Directed Co-evolution of Two Cellulosic Enzymes in Escherichia coli Based on their Synergistic Reactions

Min Liu, Lidan Ye, and Hongwei Yu

16. Program-guided Design of High-throughput Enzyme Screening Experiments and Automated Data Analysis/Evaluation

Mark Doerr and Uwe T. Bornscheuer

17. Solid Phase Agar Plate Assay for Screening Amine Transaminases

Martin S. Weiss, Uwe T. Bornscheuer, and Matthias Hoehne

18. Ultrahigh-throughput Screening of Single-cell Lysates for Directed Evolution and Functional Metagenomics

Fabrice Gielen, Pierre-Yves Colin, Philip Mair, and Florian Hollfelder

19. Isolation of pH-sensitive Antibody Fragments by Fluorescence-activated Cell Sorting and Yeast Surface Display

Christian Schroeter, Simon Krah, Jan Beck, Doreen Koenning, Julius Grzeschik, Bernhard Valldorf, Stefan Zielonka, and Harald Kolmar

20. Library Generation and Auxotrophic Selection Assays in Escherichia coli and Thermus thermophilus

Joerg Claren, Thomas Schwab, and Reinhard Sterner