Ribosome Display and Related Technologies

Display technologies are a powerful biological tool, yet the complexity of ribosome display methods has inhibited their use. This volume provides detailed and easily followed protocols with the aim of facilitating a greater uptake of these methods.

Display technologies have become a very powerful way of generating therapeutic lead molecules and specific reagents for increasing our understanding of biology; however, despite being first described shortly after phage display, the use of ribosome display and related methods have been much less widespread. Since this is in part due to the complexity of the methods, Ribosome Display and Related Technologies: Methods and Protocols seeks to extend their use by collecting expert contributions describing these detailed protocols. The protocols described range from well-established methods that have been used for a decade to generate high affinity antibodies, which are already in the clinic, to methods that are in their early stages of application such as display of peptides incorporating non-canonical amino acids. Written in the highly 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Invaluable and easy to use, Ribosome Display and Related Technologies: Methods and Protocols will be of great benefit to those with general molecular biology or protein engineering experience who wish to select peptides or proteins by display, those with phage display experience who would benefit from the application of ribosome display, as well as those with some ribosome display experience who would like to expand the range of applications to which they are applying the technology.

Features a well-balanced collection of established methods and cutting-edge techniques Includes easy to follow, step-by-step protocols, provided in clear detail Points out expert tips and implementation advice in order to ensure successful results Includes supplementary material: sn.pub/extras

Inhalt

Ribosome Display: A Perspective.- Preparation and Testing of E.coli S30 In Vitro Transcription Translation Extracts.- Eukaryotic Ribosome Display Selection Using Rabbit Reticulocyte Lysate.- Stabilized Ribosome Display for In Vitro Selection.- Eukaryotic Ribosome Display with In Situ DNA Recovery.- mRNA Display Using Covalent Coupling of mRNA to Translated Proteins.- SNAP Display: In Vitro Protein Evolution in Microdroplets.- cDNA Display: Rapid Stabilization of mRNA Display.- Optimization of Antibody Affinity by Ribosome Display Using Error-Prone or Site-Directed Mutagenesis.- Affinity Maturation of Phage Display Antibody Populations using Ribosome Display.- Evolution of Protein Stability Using Ribosome Display.- Selection of Lead Antibodies from Naive Ribosome Display Antibody Libraries.- Evolution of Disulfide-Rich Peptide Aptamers Using cDNA Display.- Peptide Screening Using PURE Ribosome Display.- Rapid Selection of High Affinity Binders Using Ribosome Display.- mRNA Display-Based Selections Using Synthetic Peptide and Natural Protein Libraries.- Identification of Candidate Vaccine Genes Using Ribosome Display.- Ribosome Display for the Selection of Sac7d Scaffolds.- Charging of tRNAs Using Ribozymes and Selection of Cyclic Peptides Containing Thioethers.- Update on Pure Translation Display with Unnatural Amino Acid Incorporation.- In Vitro Selection of Unnatural Cyclic Peptide Libraries Via mRNA Display.- Optimization of CAT-354, a Therapeutic Antibody Directed Against Interleukin-13, Using Ribosome Display.-Affinity Maturation and Functional Dissection of a Humanized Anti-RAGE Monoclonal Antibody by Ribosome Display.