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.
Part I: Review
1. Ribosome Display: A Perspective Andreas Pluckthun
Part II: Translation Extract Preparation
2. Preparation and Testing of E.coli S30 In Vitro Transcription Translation Extracts James F. Zawada
Part III: Basic Ribosome Display and Related Selection Methods
3. Eukaryotic Ribosome Display Selection Using Rabbit Reticulocyte Lysate Julie A. Douthwaite
4. Stabilized Ribosome Display for In Vitro Selection Shuta Hara, Mingzhe Liu, Wei Wang, Muye Xu, Zha Li, and Yoshihiro Ito
5. Eukaryotic Ribosome Display with In Situ DNA Recovery Mingyue He, Bryan M. Edwards, Damjana Kastelic, and Michael J. Taussig
6. mRNA Display Using Covalent Coupling of mRNA to Translated Proteins Rong Wang, Steve W. Cotten, and Rihe Liu
7. SNAP Display: In Vitro Protein Evolution in Microdroplets Miriam Kaltenbach and Florian Hollfelder
8. cDNA Display: Rapid Stabilization of mRNA Display Shingo Ueno and Naoto Nemoto
Part IV: Applications of Ribosome Display Methods Using Natural Amino Acids
9. Optimization of Antibody Affinity by Ribosome Display Using Error-Prone or Site-Directed Mutagenesis Leeanne Lewis and Chris Lloyd
10. Affinity Maturation of Phage Display Antibody Populations using Ribosome Display Maria A. Groves and Adrian A. Nickson
11. Evolution of Protein Stability Using Ribosome Display Andrew Buchanan
12. Selection of Lead Antibodies from Naive Ribosome Display Antibody Libraries Peter Ravn
13. Evolution of Disulfide-Rich Peptide Aptamers Using cDNA Display Yuki Mochizuki and Naoto Nemoto
14. Peptide Screening Using PURE Ribosome Display Hiroyuki Ohashi, Takashi Kanamori, Eriko Osada, Bintang K. Akbar, and Takuya Ueda
15. Rapid Selection of High Affinity Binders Using Ribosome Display Birgit Dreier and Andreas Pluckthun
16. mRNA Display-Based Selections Using Synthetic Peptide and Natural Protein Libraries Steve W. Cotten, Jianwei Zou, Rong Wang, Bao-cheng Huang, and Rihe Liu
17. Identification of Candidate Vaccine Genes Using Ribosome Display Liancheng Lei
18. Ribosome Display for the Selection of Sac7d Scaffolds Barbara Mouratou, Ghislaine Behar, Lauranne Paillard-Laurance, Stephane Colinet, and Frederic Pecorari
Part V: Incorporation of Non-Natural Amino Acids for Selection by Ribosome Display and Related Methods
19. Charging of tRNAs Using Ribozymes and Selection of Cyclic Peptides Containing Thioethers Patrick C. Reid, Yuki Goto, Takayuki Katoh, and Hiroaki Suga
20. Update on Pure Translation Display with Unnatural Amino Acid Incorporation R. Edward Watts and Anthony C. Forster
21. In Vitro Selection of Unnatural Cyclic Peptide Libraries Via mRNA Display Zhong Ma and Matthew C.T. Hartman
Part VI: Case Studies
22. Optimization of CAT-354, a Therapeutic Antibody Directed Against Interleukin-13, Using Ribosome Display George Thom and Ralph Minter
23. Affinity Maturation and Functional Dissection of a Humanized Anti-RAGE Monoclonal Antibody by Ribosome Display Simon E. Hufton