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Whether the pursuit is commercially motivated or purely academic, engineering a novel biological catalyst is an enticing challenge. High-resolution protein structure analysis allows for rational alteration of enzyme function, yet many useful enzyme variants are the product of well-designed selection schemes or screening strategies. Enzyme Engineering: Methods and Prools provides guidance to investigators wishing to create enzyme variants with desired properties. This detailed volume covers such topics as a simple...
Whether the pursuit is commercially motivated or purely academic, engineering a novel biological catalyst is an enticing challenge. High-resolution protein structure analysis allows for rational alteration of enzyme function, yet many useful enzyme variants are the product of well-designed selection schemes or screening strategies. Enzyme Engineering: Methods and Prools provides guidance to investigators wishing to create enzyme variants with desired properties. This detailed volume covers such topics as a simple method for generating site-specific mutations within bacterial chromosomes. It also highlights the engineering of two difference types of rare-cutting endonucleases that show great potential in gene therapy applications: The newest development is the emergence of TAL effector nucleases or TALENs. Chapters describe newly developed technologies in sufficient detail so that each method can be practiced in a standard molecular biology laboratory. Written in the 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 prools, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible Enzyme Engineering: Methods and Prools will be valuable for scientists with a budding interest in protein engineering as well as veterans looking for new approaches to apply in established discovery programs.
1. A Tripartite Fusion System for the Selection of Protein Variants with Increased Stability in vivo Linda Foit and James Bardwell
2. Determining Enzyme Kinetics via Isothermal Titration Calorimetry Neil A. Demarse, Marie C. Killian, Lee D. Hansen, and Colette F. Quinn
3. GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria Olga Paley, Giulia Agnello, Jason Cantor, Tae Hyun Yoo, George Georgiou, and Everett Stone
4. Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA Binding and Cleavage Properties Sarah K. Baxter, Abigail R. Lambert, Andrew M. Scharenberg, and Jordan Jarjour
5. TAL Effector Nuclease (TALEN) Engineering Ting Li and Bing Yang
6. In vitro Evolution of Enzymes Misha V. Golynskiy, John C. Haugner III, Aleardo Morelli, Dana Morrone, and Burckhard Seelig
7. Residue-Specific Incorporation of Unnatural Amino Acids into Proteins In vitro and In vivo Amrita Signh-Blom, Randall A. Hughes, and Andrew. D. Ellington
8. Reconstructing Evolutionary Adaptive Paths for Protein Engineering Megan F. Cole, Vanessa E. Cox, Kelsey L. Gratton, and Eric A. Gaucher
9. Oligonucleotide Recombination Enabled Site-Specific Mutagenesis in Bacteria Bryan M. Swingle
10. FX Cloning: A Versatile High-Throughput Cloning System for Characterization of Enzyme Variants Eric R. Geertsma
11. Use of Sulfolobus solfataricus PCNA Subunit Proteins to Direct the Assembly of Multimeric Enzyme Complexes Hidehiko Hirakawa and Teruyuki Nagamune
12. Gene Synthesis by Assembly of Deoxyuridine Containing Oligonucleotides Romualdas Vaisvila and Jurate Bitinaite
13. Protein Engineering: Single or Multiple Site-Directed Mutagenesis Pei-Chung Hsieh and Romualdas Vaisvila
14. Gene Assembly and Combinatorial Libraries in S. cerevisiae via Reiterative Recombination Nili Ostrov, Laura M. Wingler, and Virginia Cornish
15. Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments Sandeep Chakraborty, Renu Minda, Lipika Salaye, Abhaya M. dandekar, Swapan K. Bhattacharjee, and Basuthkar J. Rao
16. Rational Protein Sequence Diversification by Multi-Codon Scanning Mutagenesis Jia Liu and T. Ashton Cropp
17. Screening Libraries for Improved Solubility: Using E. coli Dihydrofolate Reductase as a Reporter Jian-Wei Liu and David Ollis
18. In Vitro Directed Evolution of Enzymes Expressed by E. coli in Micro-Titre Plates
Bradley J. Stevenson, Sylvia H.C. Yip, and David L. Ollis