Protein engineering is a fascinating mixture of molecular biology, protein structure analysis, computation, and biochemistry, with the goal of developing useful or valuable proteins. Protein Engineering Protocols will consider the two general, but not mutually exclusive, strategies for protein engineering. The first is known as rational design, in which the scientist uses detailed knowledge of the structure and function of the protein to make desired changes. The s- ond strategy is known as directed evolution. In this case, random mutagenesis is applied to a protein, and selection or screening is used to pick out variants that have the desired qualities. By several rounds of mutation and selection, this method mimics natural evolution. An additional technique known as DNA shuffling mixes and matches pieces of successful variants to produce better results. This process mimics recombination that occurs naturally during sexual reproduction. The first section of Protein Engineering Protocols describes rational p- tein design strategies, including computational methods, the use of non-natural amino acids to expand the biological alphabet, as well as impressive examples for the generation of proteins with novel characteristics. Although procedures for the introduction of mutations have become routine, predicting and und- standing the effects of these mutations can be very challenging and requires profound knowledge of the system as well as protein structures in general.

Part I. Design and Computational Strategies for Protein Engineering

Combinatorial Protein Design Strategies Using Computational Methods
Hidetoshi Kono, Wei Wang, and Jeffery G. Saven

Global Incorporation of Unnatural Amino Acids in Escherichia coli
Jamie M. Bacher and Andrew D. Ellington

Considerations in the Design and Optimization of Coiled Coil Structures
Jody M. Mason, Kristian M. Müller, and Katja M. Arndt

Calcium Indicators Based on Calmodulin-Fluorescent Protein Fusions
Kevin Truong, Asako Sawano, Atsushi Miyawaki, and Mitsuhiko Ikura

Design and Synthesis of Artificial Zinc Finger Proteins
Wataru Nomura and Yukio Sugiura

Monobodies: Antibody Mimics Based on the Scaffold of the Fibronectin Type III Domain
Akiko Koide and Shohei Koide

Engineering Site-Specific Endonucleases
Peter Friedhoff and Alfred Pingoud

Part II. Evolutionary Strategies for Protein Engineering

Protein Library Design and Screening: Working Out the Probabilities
Michel Denault and Joelle N. Pelletier

Protein Design by Binary Patterning of Polar and Nonpolar Amino Acids
Luke H. Bradley, Yinan Wei, Peter Thumfort, Christine Wurth, and Michael H. Hecht

Versatile DNA Fragmentation and Directed Evolution With Nucleotide Exchange and Excision Technology
Sabine C. Stebel, Katja M. Arndt, and Kristian M. Müller

Degenerate Oligonucleotide Gene Shuffling
Peter L. Bergquist and Moreland D. Gibbs.

M13 Bacteriophage Coat Proteins Engineered for Improved Phage Display
Sachdev S. Sidhu, Birte K. Feld, and Gregory A. Weiss

Ribosome-Inactivation Display System
Satoshi Fujita, Jing-Min Zhou, and Kazunari Taira

Compartmentalized Self-Replication: A Novel Method for the Directed Evolution of Polymerases and Other Enzymes
Farid J. Ghadessy and Philipp Holliger

Synthesisof 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
François-Xavier Campbell-Valois and Stephen W. Michnick

A General Method of Terminal Truncation, Evolution, and Re-Elongation to Generate Enzymes of Enhanced Stability
Jochen Hecky, Jody M. Mason, Katja M. Arndt, and Kristian M. Müller

Index