pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli

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.


Introduction
Protein N α -acetylation, or N-terminal acetylation, is one of the most common co-and posttranslational 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]. N α -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 [1]. The role of Nterminal 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 [1]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 α-synuclein (αSyn) is one of proteins for which the effects of N-terminal acetylation have been shown to be central to its physiology and pathology. αSyn is a small protein (140 aa, 14.6 kDa) ubiquitously and abundantly expressed in nervous tissue [8,9]. While its exact function is still unclear, it has long been associated with the regulation of synaptic activity and neurotransmitter release [8,10]. Most importantly, both genetic and histopathologic evidence have tied it to the pathogenesis of a class of diseases known as synucleinopathies [11], including Parkinson's Disease, the second most common progressive neurodegenerative disorder [12]. The totality of αSyn in human tissue has been shown to be N α -acetylated [13,14] and a number of studies have highlighted the role of this modification in the modulation of αSyn's lipid binding, aggregation, oligomerization and helical propensity [15][16][17][18]. This is especially important given the ongoing discussion on the structure of native αSyn in a cellular environment, which requires structural studies to be performed on a species as close as possible to the one present in nervous tissue [19].
While the expression of recombinant, N α -acetylated proteins is possible in eukaryotic hosts as yeast and insect cells, a prokaryote-compatible N α -acetylation system in bacteria (e.g. E. coli) would provide a cheaper and easier-to-use alternative. Although NATs are present in bacteria and archaea the occurrence of N-terminal acetylation is much lower and the NATs' specificity and regulation are not well-characterized [23]. One approach has been to co-express yeast NATs along with the target protein in bacteria and it has been applied successfully to most of the NATs' substrates [24,25]. While promising, this method has some shortcomings. The overexpression of both NATs and the target protein under the same inducible promoter does not ensure the proper folding and assembly of the NAT complex before the expression of the N α -acetylation target begins, which, given the co-translational nature of this modification, can lead to N α -acetylated/non-N α -acetylated mixtures [25,26]. We have thus developed an improved N α -acetylation system, pTSara-NatB, under the control of a P BAD promoter [27] and tested its performance using αSyn as a model substrate.

Materials
All materials were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise noted.
αSyn expression and purification pET21a-alpha-synuclein was a gift from the Michael J. Fox Foundation MJFF (Addgene plasmid # 51486). BL21(DE3) E. coli (New England Biolabs, Ipswich, MA) were freshly cotransformed with pET21a-alpha-synuclein and pTSara-NatB and selected on ampicillin-(amp) and chloramphenicol-(cam) supplemented LB-agar plates. Cultures were grown in LB+amp+cam and induced at an OD 600 of 0.5-0.6 with 0.2% (m/v) L-arabinose and, after 30 min, with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, or with IPTG alone at an OD 600 of 0.5-0.6). Growth was continued for 4 hrs. at 37˚C under shaking. The cell pellet, after being harvested and kept frozen at -20˚C overnight, was resuspended in 20 mM Tris buffer, 25 mM NaCl, pH 8.00, and lysed by boiling for 15 min. The supernatant of a 20-min, 20,000 x g spin of the lysate was then further processed. The sample was loaded on two 5-mL (tandem) HiTrap Q HP anion exchange columns (GE Healthcare, Pittsburgh PA), equilibrated with 20 mM Tris buffer, 25 mM NaCl, pH 8.00. αSyn was eluted from the columns with a 25-1000 mM NaCl gradient in 20 mM Tris buffer, 1 M NaCl, pH 8.00. For hydrophobic interaction chromatography αSyn peak fractions were pooled and injected on two 5-mL (tandem) HiTrap Phenyl HP hydrophobic interaction columns (GE Healthcare, Pittsburgh, PA), equilibrated with 50 mM phosphate buffer, 1 M (NH 4 ) 2 SO 4 , pH 7.40. αSyn was eluted from the columns with a 1000-0 mM (NH 4 ) 2 SO 4 gradient in Milli-Q water. αSyn peak fractions were then pooled and further purified via size-exclusion chromatography on a HiPrep Sephacryl S-200 HR 26/60 column (GE Healthcare, Pittsburgh, PA) using 50 mM NH 4 Ac, pH 7.40 as running buffer. αSyn peak fractions were pooled, aliquoted, lyophilized and stored at -20˚C.

SDS-PAGE and immunoblotting
Electrophoresis and blotting reagents were obtained from Thermo Fisher Scientific (Waltham, MA), unless otherwise noted. Samples were prepared for electrophoresis by the addition of 4x NuPAGE LDS sample buffer supplemented with 2.5% β-mercaptoethanol and denatured at 85˚C for 10 min. Samples were electrophoresed on NuPAGE Novex 4-12% Bis-Tris gels with NuPAGE MES-SDS running buffer and using the SeeBlue Plus2 MW marker. Gels were Coomassie Brilliant Blue-(CBB) stained using GelCode Blue Safe Protein Stain, according to the manufacturers' protocol, and imaged using a LI-COR Odyssey Classic scanner (LI-COR Biosciences, Lincoln, NE). After the electrophoresis, for immunoblotting, gels were electroblotted onto Immobilon-PSQ 0.2 μm PVDF membrane (Millipore, Billerica, MA) for 1 hr. at 400 mA constant current at 4˚C in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol transfer buffer. After transfer, the membranes of gels run with lysate samples were incubated in 4% (m/v) paraformaldehyde in phosphate buffered saline (PBS) for 30 min at RT, rinsed (3x) 5 min with PBS and blocked with a 5% milk solution (PBS containing 0.1% (v/v) Tween 20 (PBS-T) and 5% (m/v) powdered milk) for either 1 hr. at RT or overnight at 4˚C. After blocking, membranes were incubated in primary antibody in 5% milk solution for either 1 hr. at RT or overnight at 4˚C. Membranes were washed (3x) 5 min in PBS-T at RT and incubated (30 min at RT) in horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Pittsburgh, PA) diluted 1:10,000 in 5% milk solution. Membranes were then washed (3x) 5 min in PBS-T and developed with SuperSignal West Dura according to manufacturers' instructions.

Mass spectrometry
Samples were analyzed on an ABI 4800 TOF/TOF Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometer (Applied Biosystems, Foster City, CA). Samples undergoing trypsin digestion were incubated overnight in 50 mM NH 4 HCO 3 , 5 mM CaCl 2 , and 12.5 ngÁμL -1 of trypsin, then desalted and concentrated using Millipore C18 ZipTips before spotting. Both trypsin-digested samples and samples for intact mass analysis were prepared for spotting by mixing 0.5 μL of sample with 0.5 μL of α-cyano-4-hydroxy-trans-cinnamic acid (10 mgÁml -1 in 70% acetonitrile, 0.1% TFA). After drying, samples were rinsed with 0.1% TFA. In addition to external calibration, when measuring intact masses insulin was added as an internal standard, for higher accuracy.

Results
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 [26] and recently implemented [25] 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 [28], 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 [29] was also corrected), (Fig 1A) and called the construct pTSara-NatB. We then verified the success of the expression by CBBstained 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-alphasynuclein) 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 P BAD -regulated genes [27], was used for the induction of the ara operon. L-arabinose was added upon reach of a culture density (OD 600 ) 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).
The N-terminal acetylation efficiency of pTSara-NatB was then tested, using αSyn as a substrate, with the same protocol described before for double transformation and sequential induction. Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) Mass Spectrometry (MS) of the purified protein from BL21(DE3) E. coli co-transformed with pTSara-NatB and pET21a-alpha-synuclein, either induced or non-induced with L-arabinose, shows, somewhat surprisingly, complete substrate N α -acetylation in both cases (Fig 2). However, these results can be easily explained by the fact that complete silencing of the ara operon is not attainable by simple absence of the inducer. The catalytic nature of the NatB complex ensures that even small amounts, constitutively expressed, can acetylate efficiently the totality of the target protein. Confirming this mechanistic explanation, addition of D-glucose (0.2%) to the bacterial cultures, which has been shown to reduce the level of non-induced expression of P BAD -regulated genes through catabolite repression [27,30], reduced the fraction of N αacetylated αSyn to about 50% (S1 Fig). We also found that such mixtures of N α -acetylated and non-N α -acetylated αSyn can be resolved by hydrophobic interaction chromatography (S2 Fig). pTSara-NatB thus works as a low-level constitutive expression vector and can potentially be Larabinose-regulated in the case of difficult substrates (see, e.g., [24]).   Table 1). The~42 Da shift in the N-terminal fragment (770.43 Da ! 812.45 Da) confirms the successful and complete N-terminal acetylation of αSyn upon NatB co-expression.
https://doi.org/10.1371/journal.pone.0198715.g003 pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli Since MALDI-TOF MS could mask the presence of a small population of non-N αacetylated substrate, trypsin digestion followed by MALDI-TOF MS was also performed on a control (non-N α -acetylated) sample and one of purified αSyn from non-L-arabinose-induced co-transformed E. coli (100% N α -acetylated according to MALDI-TOF MS). The mass spectrum of the fragments (Fig 3 and Table 1) confirms the N-terminal +42 Da mass shift that corresponds to N-terminal acetylation and the efficiency of the co-translational modification (>97%).
Finally, especially given the constitutive expression of our construct, the effects of widespread N-terminal acetylation on the bacterial proteome (and in particular its potential toxicity or metabolic modulation) were tested comparing the growth curves of BL21(DE3) transformed with either pET21a-alpha-synuclein alone or pET21a-alpha-synuclein+pTSara-NatB (with or without L-arabinose induction) (Fig 4). Both in non-L-arabinose-induced (pET+-pTSara) and induced (pET+pTSara+0.2% ara) co-transformed E. coli there is an increase in the OD 600 of the cultures, when compared to those of singly transformed bacteria (pET). No toxicity is thus observed, rather an increased bacterial proliferation (although the kinetics of the growth do not appear to be changed by the introduction of N-terminal acetylation), the reason of which has not been yet investigated. It must be noted how such increased culture density does not reflect in increased yields of N α -acetylated αSyn (in contrast with what previously reported for other NatB substrates [24]).

Discussion
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 N α -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.
A clear advantage of the pTSara-NatB-mediated N-terminal acetylation is the ease which the uninduced, constitutive expression of NatB ensures. Viable substrates can be completely acetylated merely by the presence of the construct in co-transformed bacteria. In addition, the L-arabinose-inducible system (possibly in combination with a rhamnose-controlled expression vector in the case of problematic targets [25]) provides a flexibility that should secure the pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli complete N-terminal acetylation of even intractable substrates. In addition, all our testing was done in LB medium and showed excellent N α -acetylation efficiency (pNatB has been reported to perform best in rich culture media, such as NZY [26]), which could possibly extend its use to minimal medium, as in the expression of isotopically-labeled recombinant proteins. We also observed, as previously reported [26], how N-terminal acetylation is complete only in freshly transformed E. coli. Future developments should be the cloning of similar constructs for the other members of the NAT family [25], so to allow the recombinant expression of the whole N-terminal acetylome in bacteria, and extensive testing on a variety of substrates and culture conditions.