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.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

“…It was demonstrated that the native acetylation machineries in E. coli can be exploited to effectively acetylate human thymosin α1, suggesting that production of acetylated proteins is likely achievable in recombinant microbes ( Figure 1A and Table 1) [47]. Similar to the method used in protein phosphorylation, coexpression of eukaryotic acetylation enzymes, such as NatA or NatB complex from yeast, was shown to provide an effective means for amino-terminal acetylation (Table 1) [48]. Other, more complicated approaches involved site-directed incorporation of acetyllysine, an acetyl-derivative of lysine, on target recombinant protein using a three-plasmid expression system (Table 1) [49].…”

Co-translational, Post-translational, and Non-catalytic Roles of N-Terminal Acetyltransferases

“…These recent studies of a-Syn underline the importance of using correctly modified (Nt-acetylated) a-Syn in biochemical studies. Of particular interest in that regard is a recently optimized recombinant expression system for rapid production of Nt-acetylated proteins that can be used for screening potential drugs targeting Nt Ac-dependent a-Syn oligomerization (Eastwood et al, 2017).…”

Synthesis of Recombinant Human Hemoglobin With NH2‐Terminal Acetylation in Escherichia coli

“…pGM expression plasmid containing the ampicillin resistance gene and the Shine-Dalgarno (SD) sequence located between the αand β-globin genes. The pNatA plasmid contains the genes that confer chloramphenicol resistance and the naa10 and naa15 genes of the NatA complex (Eastwood et al, 2017). The pMAP expression plasmid contains the kanamycin resistance gene and an additional copy of the MAP gene (Natarajan et al, 2011).…”

“…The pNatA plasmid (pACYCduet-naa10-naa15) was a gift from Dan Mulvihill (Eastwood et al, 2017). The pGM plasmid has an ampicillin resistance gene, while pMAP and pNatA plasmids code for kanamycin and chloramphenicol antibiotic resistance genes, respectively ( Fig.…”