Ma Ying, Di Salvo Martino L, Budisa Nediljko
Biocatalysis Group, Department of Chemistry, Berlin Institute of Technology/TU Berlin, Berlin, Germany.
Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Rome, Italy.
Methods Mol Biol. 2018;1728:127-135. doi: 10.1007/978-1-4939-7574-7_7.
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).
将非标准氨基酸（ncAAs）掺入蛋白质的常见方案需要将所需的ncAA添加到生长培养基中，使其被细胞摄取，并随后在细胞内积累。这种添加方式通常适用于小规模的概念验证掺入实验。然而，这并不是ncAAs正交翻译的通用解决方案，因为它们的化学合成通常繁琐且昂贵。在这里，我们描述了一种简单的方案，该方案有效地将L-叠氮高丙氨酸的原位半合成生物合成及其在L-甲硫氨酸（Met）位置掺入蛋白质耦合在一起。在我们经过代谢工程改造的Met营养缺陷型大肠杆菌菌株中，Aha由外部添加的叠氮化钠和O-乙酰-L-高丝氨酸作为廉价前体生物合成。这代表了一个用于表达含叠氮蛋白质的有效平台，适用于旨在进行非侵入性蛋白质修饰的位点选择性生物正交策略（Tornøe等人，《有机化学杂志》67:3057 - 3064，2002；Kiick等人，《德国应用化学国际版》39:2148 - 2152，2000；Budisa，《德国应用化学国际版英文》47:6426 - 6463，2004；van Hest，《美国化学会志》122:1282 - 1288，2000）。
