Main description:

This detailed volume explores non-canonical amino acids (ncAAs) through their site-specific incorporation by genetic code expansion (GCE). The collection provides a broad resource of methods for implementing GCE in E. coli, mammalian cells, and animals, highlighting specific applications ranging from fluorescence labeling to photocontrol and the study of protein post-translational modification. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols serves as an ideal source of methodologies that can be adapted and extended, migrated to different model systems, and combined in new ways to help explore a wide range of biological questions and to augment industrial and pharmaceutical protein engineering.

Contents:

Part I: Genetic Code Expansion in E. coli and Its Applications

1. Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Non-Canonical Amino Acids

Nikolaj G. Koch and Nediljko Budisa

2. Engineering Homogeneous Photoactive Antibodies Fragments

Thomas Bridge and Amit Sachdeva

3. Repurposing Photosensitizer Proteins through Genetic Code Expansion to Facilitate Photo-Biocatalysis

Yan Xia, Xuzhen Guo, and Jiangyun Wang

4. Genetic Encoding of a Fluorescent Non-Canonical Amino Acid as a FRET Donor for Analysis of Deubiquitinase Activities

Manjia Li and Tao Peng

5. Creating Selenocysteine-Specific Reporters Using Inteins

Christina Z. Chung, Dieter Soell, and Natalie Krahn

6. Protein Expression with Biosynthesized Non-Canonical Amino Acids

Yong Wang, Wenkang Cai, Boyang Han, and Tao Liu

7. Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Non-Canonical Amino Acids into Proteins in E. coli

Han-Kai Jiang and Jeffery M. Tharp

8. Encoding Non-Canonical Amino Acids into Phage Displayed Proteins

Cristina Diaz-Perlas, Montserrat Escobar-Rosales, Charles W. Morgan, and Benjami Oller-Salvia

9. Genetically Encoded Non-Canonical Amino Acids in Proteins to Investigate Lysine Benzoylation

An-Di Guo and Xiao-Hua Chen

10. Semi-Synthesis of Glutamine-Methylated Proteins Enabled by Genetic Code Expansion

Weimin Xuan and Xiaochen Yang

Part II: Applications in Mammalian Cells

11. Genetic Code Expansion in Mammalian Cells

Zhigang Wu and Jie Wang

12. Generation of Amber Suppression Cell Lines Using CRISPR-Cas9

Birthe Meineke and Simon J. Elsasser

13. Genetic Code Expansion in Mammalian Cells through Quadruplet Codon Decoding

Yan Chen, Tianyu Gao, Xinyuan He, Wei Niu, and Jiantao Guo

14. Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation

Han Sun, Yang Huang, and Yu-Hsuan Tsai

15. Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Non-Canonical Amino Acids

Maria Kowalski-Jahn, Hannes Schihada, and Gunnar Schulte

16. Selective Inhibition of Kinase Activity in Mammalian Cells by Bioorthogonal Ligand Tethering

Jinghao Chen, Yang Huang, Wen-Biao Gan, and Yu-Hsuan Tsai

17. Site-Specific Incorporation of Sulfotyrosine into Proteins in Mammalian Cells

Xinyuan He, Yan Chen, Jiantao Guo, and Wei Niu

Part III: Genetic Code Expansion in Other Models

18. Small Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code

Wes Brown, Carolyn Rosenblum, and Alexander Deiters

19. Non-Canonical Amino Acid Incorporation in Mice

Zhetao Zheng and Qing Xia