Date Published: July 11, 2018
Publisher: Public Library of Science
Author(s): Matteo Rovere, Alex Edward Powers, Dushyant Shailesh Patel, Tim Bartels, Stephan N. Witt.
N-terminal acetylation is one of the most common co- and post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular α-synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely Nα-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely Nα-acetylate our model substrate α-synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes.
Protein Nα-acetylation, or N-terminal acetylation, is one of the most common co- and post-translational modifications of the eukaryotic proteome, with a vast majority of all N-termini (~80%) bearing this moiety. The reaction is catalyzed by a class of enzymes, N-terminal acetyltransferases (NATs), of which seven (NatA to NatF, and NatH) have to-date been discovered in humans and one (NatG) has been identified in Arabidopsis thaliana, with no human ortholog [1,2]. These enzymes mediate the transfer of an acetyl group from acetyl-CoA to the positively charged N-terminus of the protein. Their activity often requires the formation of a complex with the ribosome, mediated by one or two auxiliary, ribosome-anchoring subunits, which provide scaffolding for the catalytic subunit and, in some cases, also regulate its substrate specificity [3,4]. Nα-acetylation thus occurs usually [1,5] in a co-translational fashion, with the acetyl moiety being added to the nascent polypeptide chain [6,7]. Different enzymes of the NAT family will show different specificities for the polypeptidic substrates to be N-terminally acetylated, based on the first 2–4 amino acids of the nascent chain . The role of N-terminal acetylation varies wildly from protein to protein and organism to organism, but it has been shown to be central to protein homeostasis and cellular physiology, regulating protein half-lives, protein-protein interactions, subcellular localization, folding and aggregation .
In uncoupling the induction of the NatB complex and αSyn (or any of NatB’s substrates) two courses of action are possible: either changing the operon regulating the transcription of the NatB genes or the one acting on the SNCA (αSyn) gene. While the authors of the original NatB work suggest  and recently implemented  an N-terminal acetylation system where the target protein is under a rhamnose-inducible promoter, we decided to redesign pNatB into an arabinose-inducible system. This approach provides two clear advantages. First, using a promoter weaker than the T7/lac of the pET system will dramatically decrease the protein yield (one of the reasons for employing a bacterial expression system in the first place). In addition, the function of the N-terminal acetylation complex can be performed by catalytic amounts of enzyme and, as such, low expression levels should be more than sufficient for the complete modification of the target and, at the same time, pose less of a metabolic burden to the cells. Following the original approach used for pNatB and starting from the bicistronic construct pTSara , we cloned both the catalytic, Naa20, and regulatory, Naa25, subunit into pTSara, maintaining the ribosome-binding region of pACYC-Duet-1 (a previously reported missense A-to-G mutation in the Naa25 gene  was also corrected), (Fig 1A) and called the construct pTSara-NatB. We then verified the success of the expression by CBB-stained SDS-PAGE and immunoblotting of Naa20 and Naa25 (Fig 1B and 1C). In addition, we tested the compatibility of pTSara-NatB with the SNCA expression vector (pET21a-alpha-synuclein) by co-transforming and inducing doubly-selected cells containing both plasmids. 0.2% of L-arabinose, which has been shown to promote a robust expression of PBAD-regulated genes , was used for the induction of the ara operon. L-arabinose was added upon reach of a culture density (OD600) of about 0.5, 30 min before the addition of IPTG for pET induction. Both the expression of the NatB subunits and that of the target protein appear to be unaffected by the co-expression and there is no evidence of cross-talk (e.g. αSyn expression upon arabinose addition) between the operons (Fig 1D).
In this work we developed and characterized pTSara-NatB, an improved N-terminal acetylation construct for recombinant protein expression in E. coli. We tested its ability to completely Nα-acetylate our (model) target protein αSyn both upon L-arabinose induction and by relying only on the uninduced constitutive expression of the NatB complex subunits.