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Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns
Author(s) -
Lacoursiere Rachel E.,
O’Donoghue Patrick,
Shaw Gary S.
Publication year - 2020
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.13702
Subject(s) - ubiquitin , acetylation , lysine , genetic code , microbiology and biotechnology , chemistry , computational biology , biology , biochemistry , amino acid , gene
Ubiquitination is a post‐translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro . Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion. We also developed a protocol that optimizes acetyl‐lysine addition to minimize mistranslated proteins and maximize site‐specific acUb protein production. Purified acUb proteins were used in pilot ubiquitination assays and found to be competent with IpaH3CT and RNF8 E3 ligases. Overall, this work provides an optimized method to express and purify all acetyl‐lysine variants for ubiquitin and shows these proteins can be used to identify potential unique ubiquitination patterns.