title: An enhanced recombinant amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting α-synuclein oligomerisation.
abstract: Amino-terminal acetylation is a ubiquitous protein modification affecting the majority of eukaryote proteins to regulate stability and function. We describe an optimised recombinant expression system for rapid production of amino terminal-acetylated proteins within bacteria. We go on to describe the system's use in a fluorescence based in vivo assay for use in the high-throughput screen to identify drugs that impact amino-terminal acetylation-dependent oligomerisation. These new tools and protocols will allow researchers to enhance routine recombinant protein production and identify new molecules for use in research and clinical applications.
authors.affiliation: School of Biosciences, University of Kent, Canterbury, UK.
authors.forename: Tara A
authors.initials: TA
authors.lastname: Eastwood
authors.affiliation: School of Biosciences, University of Kent, Canterbury, UK.
authors.forename: Karen
authors.initials: K
authors.lastname: Baker
authors.affiliation: School of Biosciences, University of Kent, Canterbury, UK.
authors.forename: Holly R
authors.initials: HR
authors.lastname: Brooker
authors.affiliation: School of Biosciences, University of Kent, Canterbury, UK.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Frank
authors.affiliation: School of Biosciences, University of Kent, Canterbury, UK.
authors.forename: Daniel P
authors.identifier: http://orcid.org/0000-0003-2502-5274
authors.initials: DP
authors.lastname: Mulvihill
mesh_terms: D000107:Acetylation / Q000187:drug effects; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D055503:Protein Multimerization / Q000187:drug effects*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D015262:Xenobiotics / Q000494:pharmacology*; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D015262:Xenobiotics; D051844:alpha-Synuclein
keywords: Nat; Parkinson's disease; Tropomyosin; amino-α-acetyl-transferase; α-synuclein