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 α-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. .

Open Access

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. .