---

This volume covers some of the most widely used protocols on nanocanonical amino acids, providing details and advice for users to get each method up and running for their chosen application. Chapters have been divided into three parts describing methods for protein production in the test tube, in prokaryotes, and in eukaryotes. 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.

Authoritative and practical, Noncanonical Amino Acids: Methods and Protocols aims to provide readers with techniques that enable them to design new experiments and create new areas of research.

---

Part I: Engeneering in the Test Tube

1. Leveraging Formylglycine-Generating Enzyme for Production of Site-Specifically Modified Bioconjugates

Robyn M. Barfield and David Rabuka

2. Artificial Division of Codon Boxes for Expansion of the Amino Acid Repertoire of Ribosomal Polypeptide Synthesis

Yoshihiko Iwane, Takayuki Katoh, Yuki Goto, and Hiroaki Suga

3. Cell-free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 deletion E. coli Strains

Eiko Seki, Tatsuo Yanagisawa, and Shigeyuki Yokoyama

4. Tub-tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids

Jonas Helma, Heinrich Leonhardt, Christian P.R. Hackenberger, and Dominik Schumacher

Part II: Engeering in Prokaryotes

5. Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids

Moritz J. Schmidt and Daniel Summerer

6. Genetic Code Expansion in Enteric Bacterial Pathogens

Huangtao Zheng, Shixian Lin, and Peng R. Chen
7. Self-directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and its Incorporation into Model Proteins
Ying Ma, Martino Luigi di Salvo, and Nediljko Budisa

8. Residue-specific Incorporation of no Canonical Amino Acids for Protein Engineering

Mark B. van Eldijk and Jan C.M. van Hest
9. Using Amber and Ochre Nonsense Codons to Code Two Different Noncanonical Amino Acids in One Protein Gene

Jeffery M. Tharp and Wenshe R. Liu

10. Genetic Incorporation of Unnatural Amino Acids into Proteins of Interest in Streptomyces venezuelae ATCC 15439

Jingxuan He and Charles E. Melan‡on III

11. Expression and Purification of Site-specifically Lysine-acetylated and Natively-folded Proteins for Biophysical Investigations
Michael Lammers
12. Site-specific Incorporation of Sulfotyrosine Using an Expanded Genetic Code

Xiang Li and Chang C. Liu

13. Site-specific Protein Labeling With Tetrazine Amino Acids
Robert J. Blizzard, True E. Gibson, and Ryan A. Mehl

Part III: Engineering in Eukaryotes

14. Mapping of Protein Interfaces in Live Cells Using Genetically Encoded Crosslinkers

Lisa Seidel and Irene Coin
15. Generation of Stable Amber Suppression Cell Lines
Simon J Elsässer

16. Trapping Chromatin Interacting Proteins with Genetically Encoded, UV-Activatable Crosslinkers In Vivo
Christian Hoffmann, Heinz Neumann, and Petra Neumann-Staubitz
17. Genetically Encoding Unnatural Amino Acids in Neurons in vitro and in the Embryonic Mouse Brain for Optical Control of Neuronal Proteins

Ji-Yong Kang, Daichi Kawaguchi, and Lei Wang

18. Genetic Code Expansion-and Click Chemistry-Based Site--Specific Protein Labelling For Intracellular DNA--PAINT Imaging

Ivana Nikic

19. MultiBacTAG- Genetic Code Expansion using the Baculovirus Expression System in Sf21 Cells

Christine Koehler and Edward A. Lemke

20. Production and Chemoselective Modification of Adeno-associated Virus Site-specifically Incorporating an Unnatural Amino Acid Residue into its Capsid

Rachel E. Kelemen, Sarah B. Erickson, and Abhishek Chatterjee

21. Generation of Intramolecular FRET Probes via Noncanonical Amino Acid Mutagenesis

Simone Brand and Yao-Wen Wu

22. Fluorogenic Tetrazine--siliconrhodamine Probe for the Labeling of Non-canonical Amino Acid Tagged Proteins

Eszter Kozma, Giulia Paci, Gemma Estrada Girona, Edward A. Lemke, and Péter Kele

23. Site-specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction

Mathis Baalmann, Marcel Best, and Richard Wombacher

24. Genetic Encoding of Unnatural Amino Acids in C. elegans

Lloyd Davis and Sebastian Greiss

---