Self-Directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and Its Incorporation into Model Proteins
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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 wordsAzide Azidohomoalanine Methionine Metabolic engineering Orthogonal translation Direct sulfhydrylation pathway
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.
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