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Self-Directed in Cell Production of Methionine Analogue Azidohomoalanine by Synthetic Metabolism and Its Incorporation into Model Proteins
Citation key 176.2018.Ma
Author Ma, Y. and Salvo, M. L. and Budisa, N.
Pages 127-135
Year 2018
DOI 10.1007/978-1-4939-7574-7_7
Journal Methods. Mol. Biol.
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).
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