Advertisement

Self-Directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and Its Incorporation into Model Proteins

  • Ying Ma
  • Martino L. Di SalvoEmail author
  • Nediljko BudisaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1728)

Abstract

Common protocols for the incorporation of noncanonical amino acids (ncAAs) into proteins require addition of the desired ncAA to the growth medium, its cellular uptake, and subsequent intracellular accumulation. This feeding scheme is generally suitable for small-scale proof-of-concept incorporation experiments. However, it is no general solution for orthogonal translation of ncAAs, as their chemical synthesis is generally tedious and expensive. Here, we describe a simple protocol that efficiently couples in situ semi-synthetic biosynthesis of l-azidohomoalanine and its incorporation into proteins at l-methionine (Met) positions. In our metabolically engineered Met-auxotrophic Escherichia coli strain, Aha is biosynthesized from externally added sodium azide and O-acetyl-l-homoserine as inexpensive precursors. This represents an efficient platform for expression of azide-containing proteins suitable for site-selective bioorthogonal strategies aimed at noninvasive protein modifications (Tornøe et al., J Org Chem 67:3057–3064, 2002; Kiick et al., Angew Chem Int Ed 39:2148–2152, 2000; Budisa, Angew Chem Int Ed Engl 47:6426–6463, 2004; van Hest, J Am Chem Soc 122:1282–1288, 2000).

Key words

Azide Azidohomoalanine Methionine Metabolic engineering Orthogonal translation Direct sulfhydrylation pathway 

Notes

Acknowledgments

The authors acknowledge the financial support of the EU-funded SYNPEPTIDE (613981) consortium of FP7 and thank the Deutsche Forschungsgemeinschaft (DFG) for financial support within the research group FOR 1905. M.L.d.S. was also supported by a Research Stays Fellowship for University Academics and Scientists from Deutscher Akademischer Austauschdienst (DAAD). Y. M. acknowledges the financial support of the China Scholarship Council (CSC). We are very grateful to Dr. Hernán Biava for the preparation of Oahs.

References

  1. 1.
    Ma Y, Biava H, Contestabile R et al (2014) Coupling bioorthogonal chemistries with artificial metabolism: intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins. Molecules 19(1):1004–1022.  https://doi.org/10.3390/molecules19011004 CrossRefPubMedGoogle Scholar
  2. 2.
    Hwang B, Yeom H, Kim Y et al (2002) Corynebacterium glutamicum utilizes both Transsulfuration and direct Sulfhydrylation pathways for methionine biosynthesis. J Bacteriol 184(5):1277–1286.  https://doi.org/10.1128/JB.184.5.1277-1286.2002 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Di Salvo ML, Budisa N, Contestabile R (Dec. 2013) PLP-dependent Enzymes: a powerful tool for metabolic synthesis of non-canonical amino acids in molecular evolution and control (Molekulare Entwicklung und Kontrolle. Beilstein Symposium EdGoogle Scholar
  4. 4.
    Nölting B, Golbik R, Fersht AR (1995) Submillisecond events in protein folding. Proc Natl Acad Sci U S A 92(23):10668–10672CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Dong S, Moroder L, Budisa N (2009) Protein iodination by click chemistry. Chembiochem 10(7):1149–1151.  https://doi.org/10.1002/cbic.200800816 CrossRefPubMedGoogle Scholar
  6. 6.
    Budisa N, Steipe B, Demange P et al (1995) High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. Eur J Biochem 230(2):788–796.  https://doi.org/10.1111/j.1432-1033.1995.0788h.x CrossRefPubMedGoogle Scholar
  7. 7.
    Seidman CE, Struhl K, Sheen J, Jessen T (1997) Chapter 1, Escherichia Coli, plasmids, and bacteriophages. Introduction of plasmid DNA into cells. Curr Protoc Mol Biol 1(8):1–1.8.10.  https://doi.org/10.1002/0471142727 Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Biocatalysis Group, Department of ChemistryBerlin Institute of Technology/TU BerlinBerlinGermany
  2. 2.Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”Sapienza Università di RomaRomeItaly

Personalised recommendations