Main description:

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 Protocols 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 (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easily accessible Enzyme Engineering: Methods and Protocols 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.

Contents:

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