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. Author manuscript; available in PMC: 2021 Apr 2.
Published in final edited form as: Methods Mol Biol. 2020;2159:41–53. doi: 10.1007/978-1-0716-0676-6_4

Affinity Purification and Functional Characterization of Dynamin-Related Protein 1

Ryan W Clinton, Brianna L Bauer, Jason A Mears
PMCID: PMC8018536  NIHMSID: NIHMS1683807  PMID: 32529362

Abstract

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

Keywords: Dynamin-related protein, Mitochondrial fission, GTPase, Protein oligomerization, Electron microscopy

1. Introduction

Affinity purification is widely used to isolate recombinant proteins, and the breadth of tags reflects the variety of protein sequences with an attraction to a particular substrate. In classic studies, the inherent biochemical properties of dynamin were leveraged to isolate this protein from animal cells and tissues [1, 2]. More recently, several tags have been utilized to purify dynamin proteins with an emphasis on expressing proteins in bacterial culture to save on cost and to limit the time required for cell expansion before harvest [3–6]. In general, these efforts have been successful for both full-length and domain truncation constructs [7–10]. For full-length proteins, smaller affinity tags, including polyhistidine (6–10 amino acids) and calmodulin-binding (4 kDa) peptides (CBP), have been used to limit the amino acid length of these constructs, since bacteria struggle to express larger mammalian proteins. For smaller domain constructs, larger tags, including maltose-binding peptides (MBPs, ~50 kDa), have been used to great effect. In this chapter, we will focus on the use of the calmodulin-binding peptide to isolate dynamin-related protein 1 (Drp1). This protocol yields pure protein through a one-step column purification. However, additional steps are required to remove the CBP tag and prevent any influence from this non-native peptide sequence on Drp1 function.

In fact, we have found that the CBP tag can alter protein behavior if not removed [11]. This is not surprising since both the N- and C-terminal ends of dynamins are adjacent to the GTPase and assembly domains in these proteins. Introducing an affinity peptide in this region has the potential to disrupt the assembly and activity of dynamins. In this chapter, we demonstrate examples where non-native tags impact both Drp1 self-assembly and activity. For this reason, we remove the CBP peptide using a protease cleavage site adjacent to the N-terminal methionine at the start of the Drp1 sequence. Subsequent purification using size-exclusion chromatography (SEC) is performed to isolate highly pure Drp1.

After purification, several experiments can be performed to assess the functionality of the protein. Fortunately, Drp1 is a GTPase, so this enzymatic activity can be measured [12]. Because Drp1 self-assembly leads to enhanced GTPase activity [13], this assay also provides an indication of polymer formation in the presence of specific substrates, including nucleotides and lipids. The extent to which the protein oligomerizes can be quantified using sedimentation methods, but negative-stain electron microscopy is essential for detecting the type of polymer being formed [14]. Dynamins have several interaction interfaces, which can lead to alternate modes of oligomerization, depending on the substrate used. Interestingly, affinity tags can alter the oligomeric tendencies of Drp1, and these effects are highlighted in the following sections:

2. Materials

Prepare all buffers (CalA, CalB, and SEC Buffer) fresh for same-day use and store at 4 °C. All solutions should be prepared using deionized water.

2.1. Purification of Drp1

  1. CalA: 0.5 M L-arginine at pH 7.4, 0.3 M NaCl, 5 mM MgCl2, 2 mM CaCl2, 1 mM imidazole, and 10 mM β-Mercaptoethanol.

  2. CalB: 0.5 M L-arginine at pH 7.4, 0.3 M NaCl, 2.5 mM EGTA, and 10 mM β-Mercaptoethanol.

  3. SEC Buffer: 25 mM HEPES (KOH) at pH 7.5, 0.15 M KCl,5 mM MgCl2, and 10 mM β-Mercaptoethanol

    ∗ Note: Filter the SEC Buffer before flowing it over the column in order to remove any undissolved particulates.

  4. Calmodulin Affinity Resin.

  5. PreScission™ Protease.

  6. Luria Broth (LB).

  7. Amicon® Ultra-15 30 MWCO Centrifugal Filter.

  8. Isopropyl-β-D-thiogalactopyranoside (IPTG).

  9. Pefabloc® SC.

  10. HiLoad 16/600 Superdex 200 Prep Grade.

  11. BL21 Star™ (DE3) Chemically Competent E. coli.

  12. pCal-n-Ek-Drp1 vector.

  13. Ampicillin.

  14. Coomassie.

  15. Sonicator.

  16. Ultracentrifuge.

  17. Swinging Bucket Centrifuge.

2.2. Characterization of Drp1 GTPase Activity

  1. Assembly Buffer: 25 mM HEPES (KOH) at pH 7.5, 150 mM KCl, and 10 mM β-mercaptoethanol.

  2. Malachite Green Reagent: 1 mM Malachite Green Carbinol,10 mM ammonium, and 1 N HCl (see Note 1).

  3. Guanosine triphosphate (GTP).

  4. MgCl2.

  5. Drp1 protein.

  6. Plate reader.

  7. MaxiSorp™ Nunc-Immuno™ Strips.

  8. Heat block.

  9. Potassium Phosphate Monobasic (KH2PO4).

2.3. Quantitative Assessment of Drp1 Oligomerization by Sedimentation Analysis

  1. Purified protein in Assembly Buffer.

  2. Methyleneguanosine 5′-triphosphate (GMPPCP).

  3. Eppendorf 5424 Microcentrifuge.

  4. Laemmli Buffer.

  5. 4–20% SDS-PAGE Gel.

  6. InstantBlue™ Coomassie Dye.

  7. Gel imaging software (ImageJ or Gel Analyzer 2010, or alternative).

2.4. Qualitative assessment of Drp1 oligomerization by EM

  1. Purified protein in Assembly Buffer.

  2. Methyleneguanosine 5′-triphosphate (GMPPCP).

  3. Carbon-coated TEM grids.

  4. Parafilm.

  5. Filter paper.

  6. Uranyl acetate.

  7. High-precision tweezers.

  8. Electron Microscope—FEI Tecnai Spirit or F20.

3. Methods

3.1. Purification of Drp1

Both affinity and size-exclusion chromatography should be performed at 4 °C or in a cold room.

3.1.1. Preparation of Clarified Cell Lysate

  1. Transform BL21 Star™ (DE3) Chemically Competent E. coli with the pCal- n-EK-Drp1 vector.

  2. Inoculate 50 mL LB containing 100 g/mL ampicillin with a single colony of the BL21 Star™ (DE3) Cells containing the pCal- n-Ek-Drp1 plasmid. Grow this starter culture overnight at 37 °C with shaking at 200 rpm.

  3. The following day, inoculate 2 L fresh LB containing 100 g/mL ampicillin with 40 mL of the starter culture (i.e., a 1:50 dilution with 20 mL into each 1 L). Incubate at 37 °C with shaking at 200 rpm until an OD600 of 0.6–0.8 is achieved (see Note 2).

  4. Induce the cells with 1 mM IPTG, then shake at 200 rpm for 24 h at 18 °C.

  5. Harvest the culture by centrifugation at 4300 × g for 20 min at 4 °C and discard the resulting supernatant (see Note 3).

  6. Resuspend the resultant pellet 5 mL CalA per gram and then add 1 mM Pefabloc® SC.

  7. Lyse the cells by sonication on ice. Sonicate at 95% amplitude for 10 s on and 5 s off for a total of 5 min of sonication. Repeat the sonication an additional four times, for a total of five sonications. Rest the cells on ice for a minimum of 2 min in between each sonication (see Note 4).

  8. Pellet the cell debris by centrifugation at 150,000 × g for 1 h at 4 °C (see Note 5).

  9. Collect the clarified cell lysate (CL), or supernatant, and store it on ice or at 4 °C.

3.1.2. Affinity Chromatography

  1. Prepare the column with 4 mL Calmodulin Affinity Resin slurry (50% EtOH), which gives a column volume of 2 mL (see Note 6).

  2. Wash the resin with at least 5 column volumes CalA (i.e.,10 mL for 2 mL resin).

  3. Apply the CL to the column (Fig. 1a) and then allow it to flow through by gravity. Collect the flow-through (FT).

  4. Wash the column with 50 mL CalA and discard the wash after it passes through the column.

  5. Elute bound protein with CalB in eight 1 mL fractions (Fig. 1b).

  6. Regenerate the column by first stripping it with 15 mL CalB and then re-equilibrating it with 15 mL CalA.

  7. Repeat steps 12–15 by applying the FT to the column (see Note 7).

  8. Pool all protein-containing fractions (see Note 8). To remove the affinity tag, add 2 L PreScission™ Protease per milliliter of pooled protein and then incubate overnight at 4°C (see Note 9).

  9. Use an Amicon® Ultra-15 30 MWCO Centrifugal Filter to concentrate the pooled protein down to 5 mL or less. If the protein appears to precipitate or aggregate, place it on ice for 5 min before proceeding.

  10. Centrifuge the concentrated protein for 10 min at 10,000 × g for 20 min at 4 °C. Separate the supernatant (see Note 10).

Fig. 1.

Fig. 1

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Purification of Drp1. (a) Affinity purification followed by size-exclusion chromatography (SEC) is used to isolate purified protein. (b) A representative gel highlights the yield and purity of Drp1 following affinity purification. (c and d) Following SEC, void and peak fractions are observed, and most of the protein resides within the peak

3.1.3. Size-Exclusion Chromatography

  1. Equilibrate the column with one column volume of SEC Buffer (see Note 11).

  2. Inject the protein into the 5 mL sample loop.

  3. Separate the protein over the column at 0.5–1.0 mL/min (see Note 12). Do not exceed a pressure of 0.3 MPa.

  4. After all the sample has gone through the column, two peaks should be observed (Fig. 1c, d). Collect the fractions corresponding to the second peak, as the first peak contains very little Drp1 (see Note 13).

  5. Add glycerol as a cryoprotectant to a final concentration of 5%. Concentrate the protein down to 0.5–1.0 mL, once again, using a 30 MWCO concentrator.

  6. Split the protein into 15–20 L aliquots. Flash–freeze these aliquots in liquid nitrogen and then store at −80 °C for later use (see Note 14).

3.2. Characterization of Drp1 GTPase Activity

  1. Thaw all components, including GTP and Drp1, on ice.

  2. Prepare the phosphate standard in Assembly Buffer using Potassium Phosphate Monobasic (KH2PO4) diluted to the following concentrations: 100, 80, 60, 40, 20, 10, 5, and 0 μm. Add 20 μL of the appropriate standard to the corresponding well.

  3. Prepare a 3× GTP/MgCl2 solution in Assembly Buffer, and store it on ice until use. The final concentrations of GTP/MgCl2 should be 1 mM and 2 mM, respectively.

  4. Prepare a 2.4× solution of Drp1 in Assembly Buffer, and store it on ice until use. The final concentration of Drp1 should be 500 nM.

  5. Add 50 μL 2.4× Drp1 in triplicate to PCR tubes and then add 30 μL Assembly Buffer (see Note 15).

  6. Move PCR tubes to a 37°C heat block and start the GTP hydrolysis reaction soon after by adding 40 μL 3× GTP/MgCl2 with a multichannel pipette (see Note 16).

  7. At desired time points, quickly remove 20 μL of the reaction mixture, and add it to a well containing 5 μL 0.5 M EDTA, which will quench the reaction (see Note 17).

  8. Add 150 μL Malachite Green Reagent to each well and then read the plate at A650 (see Note 18).

  9. Plot the A650 values for the phosphate standards to create a standard curve.

  10. Use this curve to calculate the amount of phosphate released at each time point (Fig. 2a). The linear portion of this graph can be used to calculate the amount of phosphate released (ΔY) over time (ΔX) at that Drp1 concentration (Note 19), which can be reported as the kobs (Fig. 2b, see Note 20).

Fig. 2.

Fig. 2

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Affinity tags impact Drp1 activity. (a) The GTPase activity of Drp1 with different affinity tags was measured using a colorometric assay that measures phosphate generation. (b) The calculated activity rates (kobs) highlight the impact of distinct tags (CBP and GST) on Drp1 function

3.3. Quantitative Assessment of Drp1 Oligomerization by Sedimentation Analysis

  1. Prepare 2× Drp1 in Assembly Buffer. The final concentration of Drp1 should be 2–5 M.

  2. Prepare 2× GMPPCP/MgCl2 in Assembly Buffer. The final concentrations of GMPPCP/MgCl2 should be 1 mM and 2 mM, respectively (see Note 21).

  3. Combine the 2× Drp1 and 2× GMPPCP/MgCl2 in a 1:1 ratio (see Note 22).

  4. Incubate at room temperature for 1 h (see Note 23).

  5. Sediment via centrifugation at 16,100 × g for 30 min at 4 °C.

  6. Collect the supernatant fractions without disturbing the pellets, and place in separate tube. Resuspend the pellet in ice-cold Assembly Buffer (see Note 24).

  7. Add an appropriate amount of Laemmli Buffer to each sample before boiling for 10 min at 100 °C.

  8. Run samples on a 4–20% SDS-PAGE Gel.

  9. Stain the gel with InstantBlue™ Coomassie Dye (Fig. 3a).

  10. Quantify the relative amounts of Drp1 in each supernatant (Sint) and pellet (Pint) fraction using a gel imaging software, such as, ImageJ or GelAnalyzer2010 (see Note 25).

  11. To calculate the percent of protein in the pellet (Fig. 3b), divide the measured pellet band intensity by the sum of the supernatant and pellet band intensities (i.e., Pint/Sint + Pint).

Fig. 3.

Fig. 3

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Sedimentation assay can be used to quantify Drp1 oligomerization. (a) The sedimentation assay quantifies Drp1 self-assembly in the absence (−PCP) and presence (+PCP) of a non-hydrolyzable GTP analogue, GMPPCP. After centrifuging the sample, the supernatant and pellet fractions (left and right lanes, respectively) are run on a gel. (b) Imaging software was used to quantify the relative amount of Drp1 in the pellet

3.4. Qualitative Assessment of Drp1 Oligomerization by EM

  1. Prepare samples containing 2 M Drp1 in Assembly Buffer (see Note 26).

  2. Prepare carbon-coated mesh grids (see Note 27).

  3. On a piece of parafilm, add 5–10 L of sample along with two drops of 2% uranyl acetate.

  4. Place the grid, carbon-side down, onto the drop of protein sample. Allow the grid to remain on the drop for 30–60 s.

  5. Blot the grid on a piece of filter paper to remove excess sample.

  6. Wash the grid on the first drop of 2% uranyl acetate by touching the grid to the drop and then immediately blotting it on a piece of filter paper. Repeat wash.

  7. Place the grid on the second 2% uranyl acetate for 30–60 s.

  8. Remove excess stain by blotting the grid on a piece of filter paper.

  9. An electron microscope is used to image the grid (see Note 28). Self-assembly can be assessed (Fig. 4), and morphological differences can be measured qualitatively and/or quantitatively (see Note 29). If the grid will not be visualized immediately, store it in a grid box under a vacuum until use.

Fig. 4.

Fig. 4

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Negative-stain election microscopy (EM) provides a qualitative assessment of Drp1 oligomerization. In the absence of nucleotide (top panels), Drp1 forms smaller multimers that appear as crescent-shaped particles. In the presence of GMPPCP (bottom panels), tagless Drp1 self-assembles into larger spiral structures. Both affinity tags (CBP and GST) limit the formation of functional assemblies. Scale bar, 100 nm

Acknowledgement

The authors would like to acknowledge the Ramachandran lab for providing GST-tagged Drp1. Protein purification and characterization studies were performed by RWC and BLB. RWC was supported by the American Heart Association (16GRNT30950012). BLB is supported by the Molecular Therapeutics Training Grant (NIH, T32 GM008803-15). This work and JAM are supported by the NIH (R01 GM125844-01 and R01 CA208516-01A1).

Footnotes

1.

To make the Malachite Green Reagent, begin by diluting the HCl to 1 N. Then, add the solid components and mix on a stir plate until fully incorporated into solution. Use a 0.45 mm filter to remove any remaining particles that did not dissolve the solution. Store the solution at room temperature in the dark.

2.

We find that an appropriate OD is consistently obtained after 2.5 h of incubation.

3.

The pelleted cells can be subsequently stored long-term at −80°C until purification.

4.

Prior to sonication, the resuspended cells will appear cloudy, and after sonication, the solution should be noticeably more transparent. If the solution remains extremely cloudy, the sonication can be repeated an additional several times or 200 g/mL lysozyme can be added prior to sonicating.

5.

Using a Beckman Type 45 Ti rotor, the centrifugation speed will be ~101,000 × g.

6.

When adding resin to the column, washing the column, and eluting bound protein, pipette slowly in a circle around the periphery of the column in order to avoid channeling, and create and maintain an even layer of resin at the bottom of the column (alternatively, a frit can be used). Pipetting directly onto the resin can push it all to one side of the column, which could allow the protein to pass through the column without interacting with the resin.

7.

We find that applying the FT to the column and repeating steps 12–15 twice works well.

8.

The protein-containing fractions can be determined by SDS-PAGE or more quickly by taking 5 L from each fraction and adding 195 L Coomassie into corresponding wells.

9.

If you do not remove the tag, the protein can behave differently. This is highlighted in later sections characterizing the activity and functional assembly of the Drp1 protein.

10.

Traditionally, sedimentation assay has been performed using tabletop ultracentrifuges at higher speeds. We have found that a tabletop microcentrifuge is able to sediment Drp1 polymers to a similar extent.

11.

We use a HiLoad 16/600 Superdex 200 Prep Grade column to purify Drp1 and equilibrate the column with one column volume, which is approximately 120 mL.

12.

The fraction size we use is 1 mL.

13.

The first peak may be intense because of higher scatter in the detector, but we observe little protein in these fractions (Fig. 1c, d). This partitioning of peaks is important to remove unwanted larger Drp1 aggregates.

14.

If a specific stock concentration of protein is desired, an analytical assay to measure the concentration of protein can be performed prior to splitting it into aliquots. Then, the flow-through at the bottom of the concentrator from step 24 can be used to dilute the protein as needed.

15.

Instead of adding 30 μL Assembly Buffer, 4× lipid or other cofactors in Assembly Buffer can be added to the Drp1 solution and incubated for some period of time prior to the addition of GTP. Thus, the impact of lipids or partner proteins on the basal GTPase activity of Drp1 can be determined. When using a cofactor, we typically incubate it together with Drp1 for 15 min at room temperature before adding GTP.

16.

We use a thermocycler set to hold its temperature at 37 °C indefinitely as a heat block.

17.

For Drp1 without any lipids, partner proteins, or other cofactors added, the time points we normally use are 5, 10, 20, 40, and 60 min. In the presence of an agent that stimulates Drp1’s GTPase activity, the time points should be scaled down appropriately. For example, when cardiolipin-containing liposomes are added, our time points are 2, 4, 6, 8, and 10 min.

18.

The plate should be read within 10 min of adding the Malachite Green Reagent. This will help avoid excessive GTP hydrolysis due to the low pH of the Malachite Green Reagent.

19.

The rates of GTP hydrolysis and Kcat (turnover number) of Drp1 are dependent on the concentration of protein used.

20.

The presence of an affinity tag impacts the rate of hydrolysis by Drp1. In the case of the CBP tag, the rate is ~fourfold higher than the tagless protein (Fig. 2). Conversely, GST-tagged Drp1 has no apparent activity.

21.

Also prepare 2× MgCl2, without any GMPPCP, to serve as a control and measure sedimentation with the protein alone. As an alternative or in combination to GMPPCP, lipid nanotubes or liposomes can also be used to induce oligomerization and sedimentation.

22.

It is recommended that the final volume is 20 L or greater. It is hard to siphon smaller volumes without agitating the pellet.

23.

GMPPCP is a non-hydrolyzable GTP analogue. As an alternative, GTP and other analogues can be used. With GTP, the samples should be placed on ice after a defined length of incubation to stop the hydrolysis reaction. Centrifugation with this sample should be performed in a refrigerated microcentrifuge.

24.

Be careful not to disturb the pellet while removing the supernatant as the pellet may be difficult to see or invisible. In a tabletop microcentrifuge, the pellet will typically form on one side of the microcentrifuge tube. We insert the tubes into the tabletop microcentrifuge in a uniform orientation and then pipette from the opposite side of the tube from the pellet, even though the pellet cannot be seen.

25.

Oligomeric protein will be located in the pellet fraction. Note that the sedimentation values of CBP-tagged and tagless Drp1 are indistinguishable despite the difference in GTPase activities (compare Figs. 2 and 3). This highlights the need for a multifaceted characterization of purified protein.

26.

To analyze the capability of the protein to form spirals, rather than its apo-state, 1 mM GMPPCP, a non-hydrolyzable GTP analogue, and 2 mM MgCl2 can be included in the sample. Alternatively, nanotubes, liposomes, or partner proteins can also be incorporated to determine the ability of Drp1 to form polymers with these substrates. Incubate Drp1 together with these other components for 1–2 h at room temperature prior to imaging.

27.

The grids may be glow discharged or plasma cleaned before the sample is applied in order to increase their hydrophilicity.

28.

In the absence of Drp1 self-assembly, no discernible features are obvious. At higher magnification (~30,000–50,000×), a lawn of protein can be seen. Under conditions that promote Drp1 self-assembly, oligomers can be observed at lower magnification (~4000–6000×), and higher magnification imaging can identify general features of polymers (i.e., size, abundance, and helical patterns).

29.

The differences between tagged (CBP and GST) and tagless protein are difficult to discern with the protein alone (upper panels, Fig. 4). However, addition of GMPPCP promotes protein oligomerization with nucleotide binding. With the tagless protein, spiral structures are readily observed and cover the EM grid (lower left panel). CBP-tagged Drp1 forms rings or arcs (lower middle panel), but fails to form extended polymers. GST-tagged protein is assembly incompetent as no polymers are observed (lower right panel). Therefore, the affinity tags clearly impact the assembly properties of Drp1.

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