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Enzyme Engineering Methods and Protocols 1st Edition by James C Samuelson ISBN 1627032924 978-1627032926

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Enzyme Engineering Methods and Protocols 1st Edition Linda Foit Digital Instant Download

Author(s): Linda Foit, James C. A. Bardwell (auth.), James C. Samuelson (eds.)
ISBN(s): 9781627032926, 1627032924
Edition: 1
File Details: PDF, 4.60 MB
Year: 2013
Language: english
SKU: EB-4181472 Category: Tags: , ,

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Product details: 

ISBN 10: 1627032924  

ISBN 13: 978-1627032926

Author: James C Samuelson

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™ 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.

Table of contents: 

I. Protein Variant Generation & Mutagenesis

Techniques for creating diversity in protein sequences:

  • A Tripartite Fusion System for the Selection of Protein Variants with Increased Stability in vivo

  • In vitro Evolution of Enzymes

  • Residue-Specific Incorporation of Unnatural Amino Acids into Proteins In vitro and In vivo

  • Reconstructing Evolutionary Adaptive Paths for Protein Engineering

  • Oligonucleotide Recombination Enabled Site-Specific Mutagenesis in Bacteria

  • Protein Engineering: Single or Multiple Site-Directed Mutagenesis

  • Gene Assembly and Combinatorial Libraries in S. cerevisiae via Reiterative Recombination

  • Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments

  • Rational Protein Sequence Diversification by Multi-Codon Scanning Mutagenesis

II. Cloning & DNA Assembly

Methods for assembling genetic material and preparing it for expression:

  • FX Cloning: A Versatile High-Throughput Cloning System for Characterization of Enzyme Variants

  • Gene Synthesis by Assembly of Deoxyuridine Containing Oligonucleotides

III. Expression & Screening Systems

Systems used to express engineered proteins and screen for desired traits:

  • GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria

  • Screening Libraries for Improved Solubility: Using E. coli Dihydrofolate Reductase as a Reporter

  • In Vitro Directed Evolution of Enzymes Expressed by E. coli in Micro-Titre Plates

IV. DNA Targeting & Editing

Tools to modify or study DNA-protein interactions:

  • Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA Binding and Cleavage Properties

  • TAL Effector Nuclease (TALEN) Engineering

V. Enzyme Characterization

Technique for measuring enzyme activity and kinetics:

  • Determining Enzyme Kinetics via Isothermal Titration Calorimetry

VI. Protein Complex Assembly

Approaches to assemble multimeric protein complexes:

  • Use of Sulfolobus solfataricus PCNA Subunit Proteins to Direct the Assembly of Multimeric Enzyme Complexes

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Tags: James C Samuelson, Enzyme Engineering, Methods, Protocols 1st