328 p. 65 illus., 1 illus. in color. online resource
SUMMARY
Protein engineering is a fascinating mixture of molecular biology, protein structure analysis, computation, and biochemistry, with the goal of developing useful or valuable proteins. Divided into two sections, Protein Engineering Protocols reviews rational protein design strategies and directed evolutionary techniques, and their impact on protein engineering. The first section presents design and computational strategies for protein engineering focusing on designing a protein with desired properties, including examples covering a wide range of engineering techniques, such as protein-protein interactions, DNA binding, antibody mimics, and enzymatic activity. The second section on evolutionary techniques provides step-by-step instructions on library design and statistical assessment of library quality. New methods for DNA shuffling as well as different selection strategies are also presented. Following the successful Methods in Molecular Biology™ series format, this volume provides a comprehensive guide to the methods used at every stage of the engineering process. By combining a thorough theoretical foundation with detailed protocols, Protein Engineering Protocols will be invaluable to all research workers in the area, from graduate students to senior investigators
CONTENT
Design and Computational Strategies for Protein Engineering -- Combinatorial Protein Design Strategies Using Computational Methods -- Global Incorporation of Unnatural Amino Acids in Escherichia coli -- Considerations in the Design and Optimization of Coiled Coil Structures -- Calcium Indicators Based on Calmodulin-Fluorescent Protein Fusions -- Design and Synthesis of Artificial Zinc Finger Proteins -- Monobodies -- Engineering Site-Specific Endonucleases -- Evolutionary Strategies for Protein Engineering -- Protein Library Design and Screening -- Protein Design by Binary Patterning of Polar and Nonpolar Amino Acids -- Versatile DNA Fragmentation and Directed Evolution With Nucleotide Exchange and Excision Technology -- Degenerate Oligonucleotide Gene Shuffling -- M13 Bacteriophage Coat Proteins Engineered for Improved Phage Display -- Ribosome-Inactivation Display System -- Compartmentalized Self-Replication -- Synthesis of Degenerated Libraries of the Ras-Binding Domain of Raf and Rapid Selection of Fast-Folding and Stable Clones With the Dihydrofolate Reductase Protein Fragment Complementation Assay -- A General Method of Terminal Truncation, Evolution, and Re-Elongation to Generate Enzymes of Enhanced Stability
