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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Methods Enzymol. 2022 Aug 31;679:235–254. doi: 10.1016/bs.mie.2022.07.036

The preparation of recombinant arginyltransferase 1 (ATE1) for biophysical characterization

Misti Cartwright 1, Verna Van 1, Aaron T Smith 1,*
PMCID: PMC9871371  NIHMSID: NIHMS1834664  PMID: 36682863

Abstract

Arginyltransferases (ATE1s) are eukaryotic enzymes that catalyze the non-ribosomal, post-translational addition of the amino acid arginine to an acceptor protein. While understudied, post-translation arginylation and ATE1 have major impacts on eukaryotic cellular homeostasis through both degradative and non-degradative effects on the intracellular proteome. Consequently, ATE1-catalyzed arginylation impacts major eukaryotic biological processes including the stress response, cellular motility, cardiovascular maturation, and even neurological function. Despite this importance, there is a lack of information on the structural and biophysical characteristics of ATE1, prohibiting a comprehensive understanding of the mechanism of this post-translational modification, and hampering efforts to design ATE1-specific therapeutics. To that end, this chapter details a protocol designed for the expression and the purification of ATE1 from Saccharomyces cerevisiae, although the approaches described herein should be generally applicable to other eukaryotic ATE1s. The detailed procedures afford high amounts of pure, homogeneous, monodisperse ATE1 suitable for downstream biophysical analyses such as X-ray crystallography, small angle X-ray scattering (SAXS), and cryo-EM techniques.

1. Introduction

Proteins are a versatile group of biological macromolecules that are responsible for undertaking many indispensable functions within the cell. However, when constrained to only the 20 canonical amino acids, not every necessary cellular function may be fulfilled. Thus, the encoding capacity of the genome must be expanded, and such augmentation is achieved by the ability of proteins to be co- and/or post-translationally modified. Post-translational modifications (PTMs) of proteins may be highly varied and include the covalent addition of functional groups and/or reactive moieties, as well as the proteolysis or cleavage of the polypeptide (Conibear, 2020; Walsh, Garneau-Tsodikova, & Gatto, 2005). Many storied PTMs such as phosphorylation, methylation, and acetylation (Fig. 1) have been well-studied over the last several decades, and essential cellular functions have been ascribed to the fidelity of these modifications. For example, phosphorylation is a reversible PTM that is responsible for the addition or removal of a phosphate group on a protein at a hydroxyl group on amino acid side chains. This addition and its removal are both vital for regulating cellular metabolism and respiration (Humphrey, James, & Mann, 2015). As another example, acetylation is a reversible PTM that is characterized by the addition of an acetyl group (typically derived from acetyl CoA) that is attached to the N-terminus of a protein or at the side chain of lysine (Lys) residues. Acetylation has been shown to be important in gene regulation and transcription whereby the acetyl group neutralizes the positively-charged Lys allowing for the unwinding of DNA from the histone (Drazic, Myklebust, Ree, & Arnesen, 2016; Kumar, Thakur, & Prasad, 2021). Despite our understanding of these examples, several additional and important PTMs exist within the eukaryotic cell but have been historically understudied.

Fig. 1.

Fig. 1

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Post-translational modifications (PTMs) increase the complexity of the proteome. After transcription and subsequent translation, PTMs may alter protein function, oligomerization, activity, and even stabilization, thereby affording an expansion of the encoding capacity of the genome.

A lesser-studied but essential eukaryotic PTM is arginylation, which is the covalent addition of the amino acid arginine (Arg) to an acceptor protein, catalyzed by the enzyme arginyltransferase 1 (ATE1) (Balzi, Choder, Chen, Varshavsky, & Goffeau, 1990; Saha & Kashina, 2011; Tasaki, Sriram, Park, & Kwon, 2012; Van & Smith, 2020). Typically, Arg is added to an N-terminal aspartic acid (Asp), glutamic acid (Glu), or oxidized cysteine (Cys) residue of a polypeptide through the formation of a conventional peptide bond, extending the length of the polypeptide by one amino acid residue (Saha & Kashina, 2011). However, data have emerged demonstrating that arginylation may also occur at a side chain residue (typically Asp or Glu) through formation of an isopeptide bond rather than a conventional peptide bond (Wang et al., 2014). Regardless of the location of arginylation, this energy-independent process is catalyzed by ATE1s that use the high-energy aminoacylated Arg-tRNAArg (or fragments thereof) (Avcilar-Kucukgoze et al., 2020; Wang et al., 2011) as the Arg donor (Fig. 2), although the mechanism of this process is poorly understood due to a lack of ATE1 structural and biophysical information. Once arginylated, this PTM can have dramatic effects on protein structure and function.

Fig. 2.

Fig. 2

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Cartoon depiction of post-translational arginylation. ATE1 catalyzes the energy-independent transfer of the amino acid Arg (R) from the aminoacylated Arg-tRNAArg to an acceptor protein to create an arginylated protein.

Arginylation is emerging as a global regulator of eukaryotic cellular homeostasis through its degradative and non-degradative effects on the proteome. Arginylation has a major role within the N-degron pathway, a hierarchal determinant of intracellular protein half-life (Timms & Koren, 2020). The first step of the N-degron pathway is the methionine (Met) aminopeptidase-catalyzed removal of the N-terminal Met, which occurs for at least half of all proteins (Varshavsky, 2011, 2019). This process exposes what was previously the second amino acid as a primary (1°), secondary (2°), or tertiary (3°) destabilizing residue. Tertiary (3°) destabilizing residues are asparagine (Asn), Cys, or glutamine (Gln), and require chemical modifications before arginylation may occur. In animals and plants, Asn and Gln must be deamidated commonly by Ntan1 (NtN-amidase) and Ntaq1 (NtQ-amidase), respectively. Cys, however, must be oxidized to Cys sulfinic acid via Cys dioxygenases (Varshavsky, 2019); this process may also be catalyzed spuriously by reactive oxygen and reactive nitrogen species (Tasaki et al., 2012). Once a negative charge is imparted on the side chain of the now chemically-modified secondary (2°) destabilizing residue, proteins bearing Asp, Glu, or oxidized Cys can then be arginylated in a tRNA-dependent manner that is catalyzed by ATE1 (Van & Smith, 2020). Once these proteins are tagged with Arg—a primary (1°) destabilizing residue—these modified proteins may then be recognized by N-recognins, ubiquitinated, and degraded in a proteasomal-dependent manner. Examples of proteins arginylated and subsequently degraded in this manner include regulators of G-protein signaling (RGS), such as RGS4, RGS5, RGS16 and (most recently) RGS7 that are important in cardiovascular maturation as well as in the nervous system (Fina et al., 2021; Van & Smith, 2020). However, degradation is not the only fate for proteins and enzymes that have been post-translationally arginylated.

Arginylation was originally thought to be involved in only protein degradation, but recent and important research has shown that proteins may also be stabilized and oligomerize differently once arginylated (Van & Smith, 2020). Examples of some proteins that change their oligomeric state in response to arginylation include β-actin and calreticulin. Regarding calreticulin, arginylation during stress facilitates the production of stress granules, which is an important adaption in promoting cell survival (Carpio et al., 2013; Kashina, 2014). Calreticulin is a monomer before arginylation and a dimer after arginylation, which promotes a change in oligomerization that then allows the formation of disulfide bridges upon calcium depletion (Carpio et al., 2013). In ate1 knockouts of amoeba (Dictyostelium discoideum), a lack of arginylation resulted in β-actin abnormalities, indicating that actin-dependent processes such as cell adhesion and other cytoskeletal activities such as movement are ATE1-dependent (Batsios et al., 2019; Kashina, 2014). Very recently, studies have even shown that arginylation even stabilizes the human immunodeficiency virus (HIV) core during the uncoating process (Kishimoto et al., 2021). These cellular studies clearly demonstrate that ATE1-catalyzed arginylation affects proteins via non-degradative pathways as well, but the precise mechanism of action is unclear.

Despite its physiological importance, the structural and the biophysical properties of ATE1 are poorly understood. For example, there is currently no known structure of an ATE1 from any organism. Some mechanistic inferences have been garnered via comparison to functional analogs of ATE1 that are found in prokaryotes and are known as leucyl/phenylalanyl (L/F) transferases, which have been structurally characterized. L/F transferases are prokaryotic enzymes that transfer hydrophobic residues such as leucine (Leu) and phenylalanine (Phe) to the N-terminus of protein substrates (Suto et al., 2006). Structures of L/F transferases with and without tRNA analogs have been determined, and these structures have been used to suggest post-translational arginylation mechanisms (Suto et al., 2006; Van & Smith, 2020; Watanabe et al., 2007); however, these comparisons are no substitute for the structural and biophysical characterizations of a bona fide ATE1. The lack of this information prohibits the design of novel compounds to target intracellular arginylation as a therapeutic strategy.

To that end, in this chapter, we describe the recombinant expression and preparation of eukaryotic ATE1 to suitable purity and homogeneity for biophysical and structural characterizations. While ATE1 is found in virtually all eukaryotes (Jiang et al., 2020), in higher-ordered organisms, the gene may be alternatively spliced, leading to different isoforms and different intracellular localizations (Kwon, Kashina, & Varshavsky, 1999). To simplify this difficulty, in this chapter we have focused on the isolation and purification of ATE1 from Saccharomyces cerevisiae (ScATE1), which exists as a single (iso) form, but this protocol may be generally applicable to other eukaryotic ATE1s. We describe in detail a general procedure for the cloning, expression, metal-affinity purification, tag cleavage, and gel filtration of ScATE1 (Fig. 3) that reproducibly yields pure, monomeric, monodisperse ATE1 of sufficient purity for enzymatic assays, small-angle X-ray scattering, and X-ray crystallography, all of which may be used to decipher the enigmatic mechanism of post-translational arginylation.

Fig. 3.

Fig. 3

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Schematic cartoon depicting the process of ScATE1 purification described in this protocol.

2. General methods and analysis

Personal protective equipment such as safety glasses, lab coats, and gloves should be used while conducting these experiments. Additionally, adherence to all safety and security measures relevant for a biosafety level-1 (BSL-1) laboratory should be followed.

3. Construct design and protein expression

This section describes the large-scale expression of ScATE1 to suitable yields for downstream biophysical analyses. The expression construct of S. cerevisiae ATE1 (ScATE1) was based on the sequence from S. cerevisiae strain ATCC 204508 (Uniprot identifier P16639). A codon-optimized, synthetically-generated gene containing an additional Tobacco Etch Virus (TEV)-protease cleavage site (ENLYFQS) was subcloned into the pET-21a(+) expression plasmid using the NdeI and XhoI restriction sites, resulting in an encoded C-terminal (His)6 affinity tag when read in-frame for ease of purification. Experience shows that an affinity tag must not be placed on the N-terminus of ATE1. N-terminally tagged ATE1 generally fails to accumulate as a soluble protein, and any purified, N-terminally tagged protein is generally inactive, presumably due to misfolding of the N-terminal regulatory domain that houses the presence of the ATE1 [Fe—S] cluster (Van et al., 2021).

3.1. Equipment

  • Bunsen burner

  • ScATE1 gene subcloned into the pET-21a(+) expression plasmid, ca. 80–100 ng/μL concentration

  • BioRad MicroPulser Electroporator

  • Sterile 0.1 cm electroporation cuvettes

  • Sterilized pipette tips

  • Sterilized 1.5mL microcentrifuge tubes

  • Thermomixer Eppendorf Thermomixer C

  • Sterile 0.22μm pore size syringe filters

  • Disposable plastic syringes

  • Sterile 25mL serological pipettes

  • Thermo Scientific Nanodrop OneC

  • Thermo Scientific MaxQ 8000 incubator shaker

  • Thermo Scientific Lynx 6000 centrifuge

  • Sterile 50mL conical tubes

3.2. Reagents

  • Filter sterilized stock ampicillin at 1000 × concentration (0.1g/mL in distilled H2O)

  • Electrocompetent Escherichia coli C41 (DE3) expression cells

  • Recovery medium for cloning (MilliporeSigma part number CMR0002)

  • LB agar plates supplemented with ampicillin (100μg/mL)

  • 3 × 100mL sterile Luria Broth (LB): 1 g NaCl, 1 g yeast extract, 0.5 g Tris base/Tris(HCl) dissolved in 100mL distilled H2O and autoclaved

  • 12 × 1 L sterile Luria Broth (LB): 10 g NaCl, 10 g yeast extract, 5 g Tris base/Tris(HCl) dissolved in 1 L distilled H2O and autoclaved in 2 L baffled, glass trypsinizing flasks

  • 12mL 1M sterile isopropyl β-D-1-thiogalactopyranoside (IPTG): 2.86 g IPTG dissolved in 12mL distilled water and filter sterilized

  • Resuspension buffer: 100mM NaCl, 50mM Tris pH7.5, 5% (v/v) glycerol

3.3. Procedure

Day 1

  1. Thaw plasmid and recovery media.

  2. Set thermomixer to 37°C with shaking of 300RPM.

  3. Place tubes of desired electrocompetent E. coli cell line on ice for 15min.

  4. Add 2μL of plasmid to the cells under the flame of a Bunsen burner.

  5. Under flame of a Bunsen burner, mix and transfer the plasmid/cell mixture to a sterile, 0.1cm electroporation cuvette, ensuring the mixture is pipetted fully into the cuvette slot (see Note 1).

  6. Immediately place the covered cuvette in the electroporator and pulse using the “EC1” program.

  7. After pulsed, remove the cuvette from the electroporator. Under the flame of a Bunsen burner, add 500μL of cold recovery media to the cuvette and gently pipette up and down to mix (the mixture should appear cloudy).

  8. Still under the flame of a Bunsen burner, transfer this mixture to a new, sterile 1.5mL tube.

  9. Place the tube in the Thermomixer and mix for 30min at 300RPM.

  10. Warm an LB ampicillin agar plate at room temperature and label.

  11. After 30min, remove the tubes containing the transformed cells from the thermomixer. Under the flame of a Bunsen burner, pipette 80μL onto the LB ampicillin agar plate and spread using a sterilized spreader.

  12. Allow colonies to grow overnight by placing in a warm room or 37°C incubator.

Day 2

  1. Under the flame of a Bunsen burner and using a sterile pipet tip, pluck a single colony from the LB ampicillin agar plate and inoculate 3 × labeled, sterile flasks charged with 100mL of sterile Luria Broth (LB) supplemented with 100μL of 0.1 g/mL filter sterilized ampicillin.

  2. Place these flasks into the incubator shaker, and grow the E. coli overnight with shaking of 200 RPM at 30°C.

  3. Seal the plate with parafilm and store at 4°C.

Day 3

  1. Under the flame of a Bunsen burner, add 1mL of 0.1 g/mL filter sterilized ampicillin to each of 12 baffled, glass trypsinizing flasks charged with 1 L of autoclaved LB.

  2. Retrieve the 3× 100mL culture flasks from the incubator shaker and combine the cultures under the flame of a Bunsen burner.

  3. To each of the 12 flasks charged with 1L of autoclaved LB and supplemented with 100μg/mL, inoculate with approximately 25mL of the combined culture from step 2 (see Note 2).

  4. Allow the bacteria to grow with shaking of 200RPM in an incubator shaker set to 37°C until the cultures reach an optical density (OD) at 600nm (OD600) of ca. 0.6–0.8.

  5. Remove the flasks from the incubator shaker and cold shock the cultures by placing them at 4°C for approximately 2h.

  6. Approximately 30min prior to the end of step 5, set an incubator shaker to 18°C.

  7. Once step 5 is finished, to each of the 12 flasks induce expression by adding 1mL of 1M filter sterilized IPTG.

  8. Allow induction to occur with shaking of 200RPM in an incubator shaker set to 18°C overnight (typically 16–20h).

Day 4

  1. Harvest cells by spinning at 5000 × g, for 12min at 4°C in 4× 1L centrifuge bottles and discard the supernatant.

  2. Repeat step 1 three times until all cells have been harvested and all supernatant has been discarded.

  3. Pour off the remaining supernatant.

  4. Resuspend cells in approximately 35mL of resuspension buffer per 3 L of culture.

  5. Transfer cellular resuspension to 50mL conical tubes, flash-freeze on N2(l) and store at −80°C (see Note 3).

3.4. Notes

  1. If the cell-plasmid mixture does not go into the slot, gently tap the cuvette to allow the mixture to get to the bottom of the cuvette.

  2. A glycerol stock can be made from the remaining cells and stored in −80°C for future cell growths.

  3. The freezing step is optional. It is possible to go directly to cell lysis and purification without the intermediate freezing step.

4. Purification of (His)6-tagged ATE1

This section describes the initial purification of ScATE1 by utilizing the C-terminal (His)6 tag, which allows for the protein to be purified with immobilized metal affinity chromatography (IMAC). As previously noted (vide supra), our experience shows that the location of the ATE1 affinity tag is important, and properly folded and enzymatically active ATE1 is best achieved when the affinity tag is present on the C-terminus of the protein. After cell lysis and centrifugation, the clarified lysate is applied to the nickel-containing resin, and purification is achieved using a fast protein liquid chromatography (FPLC) instrument, which results in good purity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high yields (average of approximately 25mgL−1 culture).

4.1. Equipment

  • QSonica Q700 ultrasonic cell disruptor operating at 4°C

  • Stainless-steel beaker

  • Polycarbonate ultracentrifuge tubes

  • Beckman-Coulter Optima XE-90 ultracentrifuge

  • ÄKTA™ Pure protein purification system

  • HisTrap™ HP 5mL pre-packed protein purification column

  • Amicon® Ultra-15 centrifugal filter unit with a 30kDa molecular weight cut off (MWCO) spin filter

  • Eppendorf 5810R centrifuge

  • Gel electrophoresis equipment

4.2. Reagents

  • Wash buffer: 50mM Tris pH8.0, 300mM NaCl, 10% (v/v) glycerol, 1mM tris(2-carboxyethyl)phosphine (see Note 1)

  • Elution buffer: 50mM Tris pH8.0, 300mM NaCl, 10% (v/v) glycerol, 1mM tris(2-carboxyethyl)phosphine, 300mM imidazole

  • 15% (m/v) SDS-polyacrylamide protein electrophoresis gels

  • Solid phenylmethylsulfonyl fluoride (PMSF)

4.3. Procedure

  1. If the ATE1-containing cells were frozen in the optional part of Step 3 (vide supra), thaw cells gently to room temperature and transfer to a stainless-steel beaker.

  2. To the thawed, homogenized cells, add ca. 50–100mg of solid PMSF and stir vigorously prior to lysis (see Note 2).

  3. Immerse the stainless-steel beaker in an ice-water slurry. Sonicate for ca. 10–12min at 80% amplitude, alternating 30s on sonication pulse and 30s off sonication pulse.

  4. Transfer the lysate to polycarbonate ultracentrifuge tubes. Centrifuge at 163,000×g for 1h at 4°C to pellet insoluble cellular debris.

  5. After ultracentrifugation, decant the supernatant into a cooled beaker.

  6. Prepare your buffers, column, and sample inlet on your FPLC (see Note 3).

  7. Pre-wash the column with 5 column volumes (CVs) of a buffer mixture containing 93% (v/v) wash buffer and 7% (v/v) elution buffer.

  8. Apply your sample lysate to the column.

  9. Wash the column again with 8 CVs of a buffer mixture containing 93% (v/v) wash buffer and 7% (v/v) elution buffer.

  10. Elute any non-specifically bound proteins by washing the column with 6 CVs of a buffer mixture containing 90% (v/v) wash buffer and 10% (v/v) elution buffer.

  11. Elute ATE1 by washing the column with 6 CVs of 100% elution buffer.

  12. Pool together the elution fractions from Step 11.

  13. Wash an Amicon Ultra-15 centrifugal filter unit with a 30kDa MWCO spin filter with distilled H2O by centrifuging at 5000×g for 5min at 4°C.

  14. Discard the H2O from the tube and apply 14mL of lysate to the spin concentrator. Spin the concentrator at 5000×g for ca. 10–20min at 4°C (see Note 4).

  15. Resuspend the protein gently in the spin filter and add more elution fractions until the protein has been concentrated to ca. 1–1.5mL (typically ca. 20–40mg/mL in our hands).

  16. Remove the protein from the filter and transfer to a clean, labeled 1.5mL microcentrifuge tube.

  17. To determine the purity of the protein, apply 1–5μg of purified protein per well to a 15% (m/v) SDS-polyacrylamide gel.

  18. Aliquot the protein into fractions of desired sizes (ca. 10–1000μL), flash-freeze on N2(l) and store at −80°C (see Note 5).

4.4. Notes

  1. In our experience, it is imperative to include some sort of reducing agent in the recombinant preparation of ATE1 in order to prevent protein aggregation. Other reducing agents, such as dithiothreitol (DTT) and/or β-mercaptoethanol (BME) are suitable but will oxidize more rapidly than tris(2-carboxyethyl)phosphine.

  2. If proteolysis is observed, other inhibitor cocktails may be substituted for PMSF. However, we have not noted a need for these alternative protease inhibitors in our hands.

  3. While an FPLC makes the IMAC purification procedure more automated, it is unnecessary, and a loose resin in a gravity column may be substituted for this portion of the protocol.

  4. Depending on protein yield, concentration may be slower or quicker than reported here. This step may need to be optimized for each ATE1 construct.

  5. Freezing of the protein is optional at this step. One may go directly to tag cleavage and/or size-exclusion chromatography at this step.

5. Cleavage of (His)6-tagged ATE1

This section describes the removal of the C-terminal (His)6 tag using the Tobacco Etch Virus (TEV) protease cleavage site (ENLYFQS) that was genetically added to the construct (see Section 3). The removal of the C-terminal (His6) tag is optional, but removal of this tag may be useful for downstream applications, although in our hands, we have not noted a difference in the behavior of ATE1 with and without its C-terminal tag. After purification by an affinity column, the buffer of the purified enzyme is modified, and the ATE1 is then incubated with TEV-protease (commercial or house-made). Once cleavage has occurred, the solution is then applied to a nickel-containing resin, and the cleaved protein now elutes during the column-washing step instead of the elution step. The cleavage may be confirmed via Western blotting using an anti-(His)6 antibody (see Note 1).

5.1. Equipment

  • ÄKTA™ Pure protein purification system

  • HisTrap™ HP 5mL pre-packed protein purification column

  • Amicon® Ultra-15 centrifugal filter unit with a 30kDa molecular weight cut off (MWCO) spin filter

  • Eppendorf 5810R centrifuge

  • Gel electrophoresis equipment

5.2. Reagents

  • Wash buffer: 50mM Tris pH8.0, 300mM NaCl, 10% (v/v) glycerol, 1mM tris(2-carboxyethyl)phosphine

  • Elution buffer: 50mM Tris pH8.0, 300mM NaCl, 10% (v/v) glycerol, 1mM tris(2-carboxyethyl)phosphine, 300mM imidazole

  • 15% (m/v) SDS-polyacrylamide protein electrophoresis gels

  • Ethylenediaminetetraacetic acid (EDTA)

  • Dithiothreitol (DTT)

  • Tobacco Etch Virus (TEV) protease (commercial or house-made)

5.3. Procedure

  1. Measure the concentration of purified ATE1 from Section 4.

  2. Dilute ATE1 protein to a concentration of approximately 1mg/mL.

  3. To the protein solution, add EDTA to a final concentration of 0.5mM.

  4. To the protein solution, add DTT to a final concentration of 100mM.

  5. To the protein solution, add TEV protease to the protein in a 1:100 (w/w) ratio.

  6. Incubate the ATE1 and protease solution overnight at 4°C while constantly rocking (see Note 2).

  7. After tag cleavage, buffer exchange the ATE1 and protease mixture into wash buffer using an Amicon Ultra-15 centrifugal filter unit with a 30kDa MWCO spin filter. Spin the concentrator at 5000×g for ca. 10–20min at 4°C until the mixture is concentrated to ca. 1–2mL. Dilute the concentrated mixture with fresh wash buffer to approximately 14mL and centrifuge again until the mixture is concentrated to ca. 1–2mL. Repeat this process again until you have buffer exchanged your ATE1 and protease mixture 3–4 times.

  8. Prepare your buffers, column, and sample inlet on your FPLC.

  9. Pre-wash the column with 6 CVs of a buffer mixture containing 93% (v/v) wash buffer and 7% (v/v) elution buffer.

  10. Apply your ATE1 and protease sample solution to the column (see Note 3).

  11. Wash the column again with 8 CVs of a buffer mixture containing 93% (v/v) wash buffer and 7% (v/v) elution buffer. Make certain to collect these fractions, as your cleaved ATE1 should elute at this stage.

  12. Elute any non-specifically bound proteins by washing the column with 6 CVs of a buffer mixture containing 90% (v/v) wash buffer and 10% (v/v) elution buffer (see Note 4).

  13. Elute any protein still stuck to the column by washing the column with 6 CVs of 100% elution buffer.

  14. Separately pool together the elution fractions from Steps 11, 12, and 13.

  15. To determine the location of ATE1, apply 1–5μg of purified protein per well to a 15% (m/v) SDS-polyacrylamide gel.

  16. Once the location of cleaved ATE1 is identified, concentrate the protein to ca. 1–1.5mL (typically ca. 20–40mg/mL in our hands).

  17. Aliquot the protein into fractions of desired sizes (ca. 10–1000μL), flash-freeze on N2(l) and store at −80°C (see Note 5).

5.4. Notes

  1. While Western blotting is an easy and convenient method for determining the presence or absence of the (His)6 tag, other methods such as mass spectrometry may be used instead.

  2. The temperature and time for incubation of TEV protease with ATE1 may need to be optimized. For some constructs, room (approximately 25°C) or elevated (approximately 37°C) temperatures and shorter incubation times may be necessary to accomplish complete cleavage.

  3. If you have concentrated your sample to ca. 1–2mL, you may use a small sample loop to apply your mixture to your column (vide infra). For larger volumes, and external sample line or a superloop may be necessary.

  4. Because ATE1s are also Cys-rich proteins, you may experience some modest binding of the cleaved protein to Ni-containing resin even in the absence of the (His)6 tag. We recommend keeping all of your elution fractions and testing them to see whether your cleaved protein is present.

  5. Freezing of the protein is optional at this step. One may go directly to size-exclusion chromatography at this step.

6. Size-exclusion purification of ATE1

This section describes the final purification (or “polishing”) of ScATE1 via size-exclusion chromatography. This process may be performed with either the (His)6-tagged or the cleaved form of the protein. In both cases, the protein will be both further purified and buffer exchanged into a more suitable downstream buffer, while in the latter case, this process of gel filtration will also remove the small amount of TEV protease present in the cleavage mixture. Moreover, this process affords the separation of differing oligomeric states of ScATE1 and allows for the removal of any aggregated protein that may have been present during purification and/or created due to spurious oxidation of the O2-sensitive ScATE1. Finally, using calibration standards for the column of interest, this process reveals that ScATE1 is a predominantly monomeric protein (regardless of the presence or absence of the (His)6 tag), which is the first time this observation has been reported, to our knowledge. This procedure is broadly applicable to other eukaryotic ATE1s, and the final purified protein is suitable for structural and biophysical characterizations.

6.1. Equipment

  • ÄKTA™ Pure protein purification system

  • Eppendorf 5418R microcentrifuge

  • 3mL syringes

  • BD PrecisionGlide™ 20G needle

  • ÄKTA™ sample loop, 2.0mL

  • HiLoad™ 16/600 Superdex 200pg preparative gel filtration column pre-calibrated at 4°C

  • Sterile 0.22μm bottle top filters for 45mm media bottles

  • Schlenk line

  • Schlenk line adapter top for 45mm media bottles

  • Stir plate

  • Stir bar

  • 1.5mL microcentrifuge tubes

  • Sterile 1.5mL microcentrifuge tubes containing 0.22μm filters

  • Agilent Cary 60 UV–Visible spectrophotometer

  • UV-transparent 1cm cuvette

6.2. Reagents

  • Size-exclusion buffer: 50mM Tris pH7.5, 100mM KCl, 5% (v/v) glycerol, 1mM dithiothreitol (DTT) (see Note 1)

6.3. Procedure

Day 1

  1. Prepare the size-exclusion buffer and filter through a sterile 0.22μm bottle top filter.

  2. Add a stir bar to the buffer, attach the Schlenk line adapter to the bottle, and connect the adapter to the Schlenk line vacuum.

  3. With stirring, degas the buffer for at least several hours, typically overnight (see Note 2).

Day 2

  1. Pack the outside of the degassed buffer bottle with ice to cool the buffer.

  2. If the purified ATE1 were frozen in the optional part of Step 4 or Step 5 (vide supra), thaw the protein (ca. 10–20mg) gently on ice.

  3. Once thawed, filter the protein briefly by centrifuging at 5000×g for 5min at 4°C through a 1.5mL microcentrifuge tube containing 0.22μm filter in order to remove any insoluble material.

  4. Attach the 2mL sample loop to the FPLC, connect to a clean 3mL syringe, and rinse the sample loop several times with size-exclusion buffer.

  5. Using a 20G needle attached to a 3mL syringe, load the purified ATE1 into the syringe, invert, and remove any bubbles.

  6. Remove the needle carefully and attach the syringe to the sample loop to load the purified ATE1 into the sample loop (see Note 3). Once loaded, finish attaching the sample loop to the FPLC.

  7. Pre-equilibrate the size-exclusion column with 1.5 CVs of cold, degassed size-exclusion buffer.

  8. Apply the sample in the 2mL sample loop to the column by washing the sample loop with 6mL of cold, degassed size-exclusion buffer.

  9. Elute the protein isocratically from the size-exclusion column by applying another 1.5 CVs cold, degassed size-exclusion buffer. For a column of this size, we typically collect protein in 1 or 2mL fractions, but the fraction size can be optimized for each construct, depending on its oligomeric homogeneity.

  10. Based on the elution volume and standards that were previously calibrated on the size-exclusion column, calculate the estimated oligomeric state of each elution peak. If it is unclear which peak is your protein of interest, apply 1–5μg of purified protein per well to a 15% (m/v) SDS-polyacrylamide gel to determine which fractions to pool.

  11. If you have multiple elution peaks containing ATE1, pool and concentrate each in separate Amicon Ultra-15 centrifugal filter units each containing a 30kDa MWCO spin filter. Our preparations are typically dominated by a single, monomeric elution peak (Fig. 4), which is generally the only protein form that we pool and concentrate.

  12. Once concentrated to the desired concentration (typically 10mg/mL for our purposes), apply 1–5μg of purified protein per well to a 15% (m/v) SDS-polyacrylamide gel to determine the final purity of the protein (Fig. 4).

  13. Estimate the total amount of protein retrieved by measuring the protein absorbance at 280nm (A280) and calculating the concentration using an estimated molar absorptivity (ε) of 87,700M−1 cm−1 for apo ScATE1 (see Note 4).

  14. Aliquot the protein into fractions of desired sizes (ca. 10–1000μL), flash-freeze on N2(l) and store at −80°C (see Note 5).

Fig. 4.

Fig. 4

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Following the protocol described in this chapter, recombinantly expressed and purified ScATE1 is homogeneous and monodisperse. (A) Superdex 200 size-exclusion chromatogram of post-IMAC-purified ScATE1. Based on calibration standards, ScATE1 is predominantly monomeric under these conditions. (B) 15% SDS-PAGE analysis of IMAC- and SEC-purified ScATE1. The left lane is the molecular weight (MW) marker, while the right lane is purified ScATE1. The left arrow points to purified ScATE1 with an apparent molecular weight of ≈60 kDa.

6.4. Notes

  1. The size-exclusion buffer composition can require trial-and-error testing. In general, for structural studies, lower salt concentrations are preferred. However, not all ATE1 constructs tolerate lower salt concentrations. In lieu of DTT, tris(2-carboxyethyl)phosphine or BME are acceptable substitutes, but their oxidation times are different than DTT.

  2. This step is important to prevent bubbles from disrupting the gel-filtration matrix. The buffer should be degassed until no more bubbles are visibly observed while stirring under vacuum, although the precise length of time may differ based on buffer composition.

  3. The exact amount of protein that may be applied to the size-exclusion column will differ from column to column and protein to protein. Generally, we apply no more than 10–20mg per run for HiLoad™ 16/600 Superdex 200pg, but we have applied up to 100mg of protein in some cases with only minimal loss of peak-to-peak resolution.

  4. The molar absorptivity presented is calculated theoretically but is generally accurate in our experience if the protein is apo, i.e., lacks the [Fe—S] cluster.

  5. Freezing of the protein is optional at this step, and the protein may be used directly for downstream applications.

7. Summary and conclusions

This chapter describes the recombinant expression and preparation of a eukaryotic ATE1 to suitable purity and homogeneity for downstream biophysical and structural characterizations. Understanding the mechanism of ATE1-catalyzed post-translation arginylation is paramount, as it is well known that ATE1 is a critical enzyme for a variety of biological processes including cellular homeostasis, cardiovascular development, and cellular motility. Moreover, recent research has shown that ATE1 is linked to several human diseases, such as cancer and HIV. However, despite this importance, there is currently no published structure of an ATE1 from any organism, and there is very little published research on any biophysical or enzymatic characterization of this important enzyme, which is a major roadblock in efforts to understand the mechanism of this emerging post-translational modification. The protocol detailed in this chapter focuses on efforts to express S. cerevisiae ATE1 (ScATE1) and to purify this ATE1 using chromatographic methods. The data presented here clearly show that this approach delivers large quantities of monomeric and monodisperse ScATE1. Based on size-exclusion data, we also show that ScATE1 behaves monomerically under these conditions, which was inferred but not known previously. This well-behaved protein can be used for downstream biophysical and biochemical studies, which should result in a better understanding of the structure, the mechanism, and the enzymatic properties of ScATE1. Additionally, the procedure outlined in this chapter may be generally applied to other eukaryotic ATE1s allowing for the isolation and purification of enzyme from other organisms, which is currently underway. Ultimately, a greater understanding of the structure and the function of ATE1 as well as the mechanism of post-translation arginylation should lead to further research to allow the targeting of this PTM for therapeutic purposes.

Acknowledgments

This work was supported by NIH-NIGMS grant R35 GM133497 (A.T.S and V.V.) and in part by NIH-NIGMS supplement R35 GM133497-03S1 (M.C.).

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