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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.
List of contents
Protein Engineering - Past, Present, and Future.- (Semi)rational Protein Design.- Computational Analysis of Protein Tunnels and Channels.- YASARA - A Tool to Obtain Structural Guidance in Biocatalytic Investigations.- A Computational Library Design Protocol for Rapid Improvement of Protein Stability - FRESCO.- Directed Evolution of Proteins Based on Mutational Scanning.- A Brief Guide to the High-throughput Expression of Directed Evolution Libraries.- Library Growth and Protein Expression: Optimal and Reproducible Microtiter Plate Expression of Recombinant Enzymes in E. coli using MTP Shakers.- Normalized Screening of Protein Engineering Libraries by Split-GFP Crude Cell Extract Quantification.- Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.- Practical Considerations Regarding the Choice of the Best High-throughput Assay.- High-throughput Screening Assays for Lipolytic Enzymes.- Continuous High-throughput Colorimetric Assays for -transaminases.-Colorimetric High-troughput Screening Assays for the Directed Evolution of Fungal Laccases.- Directed Co-evolution of Two Cellulosic Enzymes in Escherichia coli Based on their Synergistic Reactions.- Program-guided Design of High-throughput Enzyme Screening Experiments and Automated Data Analysis/Evaluation.- Solid Phase Agar Plate Assay for Screening Amine Transaminases.- Ultrahigh-throughput Screening of Single-cell Lysates for Directed Evolution and Functional Metagenomics.- Isolation of pH-sensitive Antibody Fragments by Fluorescence-activated Cell Sorting and Yeast Surface Display.- Library Generation and Auxotrophic Selection Assays in Escherichia coli and Thermus thermophiles.
Summary
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.
