Method Article

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

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DOI:

10.3791/54860

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December 17th, 2016

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1Department of Biology, Indiana University-Purdue University, Indianapolis (IUPUI)

In This Article

Summary

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Abstract

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric β subunit rings sandwiched between two heptameric α subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome α and β subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete α-ring and one complete β-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

Introduction

Multiprotein complexes carry out numerous critical cellular activities1. For many of these complexes, much more is known about their structure and function than about their assembly2,3. The proteasome is one such complex and is found in all domains of life. In eukaryotes, this molecular machine is at the core of the Ubiquitin/Proteasome System (UPS) and provides the major route of intracellular protein degradation4. The eukaryotic proteasome (referred to as the 26S proteasome) is comprised of two major sub assemblies: a 20S proteasome, or Core Particle (CP)5, that can be capped on one or both ends by a 19S Regulatory Particle (RP)6.

The 20S proteasome is a large compartmentalized protease. Its quaternary structure is absolutely conserved across all domains of life and consists of a stack of four seven-membered rings containing two types of structurally related subunits, α and β5,7,8. In eukaryotes, the two outer rings are each comprised of seven distinct α subunits and the two inner rings are each comprised of seven distinct β subunits; proteolytic activity resides within three of the β subunits. By contrast, the CP rings of archaea and bacteria are usually comprised of only one type of α and one type of β subunit. Archaeal proteasomes have provided an important model system to study proteasome assembly due to both their compositional simplicity and their sharing a common assembly mechanism with their eukaryotic counterparts9-13. In brief, α subunits assemble into α rings first, which serve as a scaffold onto which β subunits assemble. The resulting half-proteasomes (α7β7) dimerize, giving rise to fully assembled CP (α7β7β7α7). During dimerization, the propeptides present on β subunits are autocatalytically removed, exposing the catalytic N-terminal threonines. The use of archaeal proteasomes to model assembly frequently takes advantage of the production of recombinant archaeal proteasome proteins in Escherichia coli. This is a worthwhile approach because it enables the subunits to be produced in various combinations, as both WT and mutant versions, in a host organism that does not produce its own proteasomes.

Monitoring the assembly of multi-protein complexes biochemically requires some kind of fractionation method that separates fully assembled complexes from assembly intermediates and precursors. Due to its superior resolving capacity, nondenaturing Polyacrylamide Gel Electrophoresis (PAGE) has proven to be especially useful in the fractionation of various large multiprotein complexes14-17. The combination of recombinant archaeal proteasome production and nondenaturing PAGE has become a powerful approach in dissecting proteasome assembly9,11,12,18. However, the usual method by which this approach is applied (i.e. via the recombinant coexpression of α and β subunits) has an important drawback. Assembly reactions are cooperative and strongly concentration-dependent3. Given that the protein concentration inside cells is very high19, due to excluded volume effects, assembly reactions proceed rapidly in vivo. Hence it is possible to miss important assembly intermediates when α and β subunits are coexpressed.

Here, we argue for a combined approach in the study of proteasome assembly using recombinant archaeal proteasome subunits. In this approach, both coexpression and lysate mixing methods are employed. The former allows for rapid analysis of assembly because coexpression is less labor intensive. The latter depends on separate expression of α and β subunits, followed by mixing. Though this requires a bit more effort than coexpression, it is more than compensated for by the ability to detect intermediates that are missed during coexpression. Together, these two methods can provide a more complete picture of proteasome assembly.

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Protocol

1. Bacterial Expression.

Note: Expression plasmids used in this study are described in Table 1. Solutions, media, and buffers used in this study are described in Table 2. The cloning of archaeal proteasome subunit genes and the generation of expression plasmids are described elsewhere18,20. In brief, plasmids for recombinant coexpression of subunits employ a bicistronic operon strategy which helps in obtaining comparable expression levels of individual subunits18,20. The expression parameters listed below were empirically determined to be optimal for the proteasome subunits in this study. It may be necessary to optimize expression for other proteasome mutants, for proteasomes from other archaeal species, or for other recombinant protein complexes (see Discussion).

  1. Transform expression plasmid of interest into chemically competent E.coli BL21(DE3) cells.
  2. Add 1 - 2 μL of plasmid (typically 50 - 100 ng) to an aliquot of DE3 cells that were freshly thawed on ice, and continue to incubate on ice for 20 min.
  3. Heat shock the cells at 42 °C for 45 s and return tube to ice for additionl 2 min.
  4. Add 1 mL of LB medium and incubate at 37 °C with shaking (150 rpm) for 1 h.
  5. Spread 100 - 200 μL of the transformation mixture onto LB-kan plates (plates containing solid LB media, supplemented with kanamycin (all plasmids used in this study encode resistance to this antibiotic)). Incubate plates at 37 ºC O/N.
  6. Next morning, inoculate a single colony from the LB-kan plate into 3 mL of liquid LB-kan media in a glass culture tube. Incubate at 37 ºC with shaking (150 rpm) for approximately 2.5 - 3 h, or till turbidity is observed.
  7. Measure the optical density of the culture at 600 nm (OD600) in a spectrophotometer. Dilute the cell culture with an appropriate amount of prewarmed LB-kan media to an OD600 of 0.4 in a final volume of 6 mL. Resume shaking at 37 ºC for 40 min.
  8. Induce protein expression in the liquid cultures by adding IPTG from a stock solution to a final concentration of 1 mM. Incubate at 37 ºC with shaking (150 rpm) for approximately 6 - 7 h.
  9. Harvest bacterial cell cultures in their entirety into 1.5 mL microcentrifuge tubes.
  10. Add 1.5 mL of a culture into microcentrifuge tube. Centrifuge at 10,000 × g for 1 min.
  11. Discard the supernatant and add another 1.5 mL of the same culture to the pellet. Centrifuge at 10,000 × g for 1 min.
  12. Repeat step 1.11 until the entire culture is harvested into the microcentrifuge tube.
  13. Store the induced pellets at -80 °C until lysis.

2. Bacterial Lysis and Lysate Mixing.

Note: For samples studied via coexpression, follow section 2.1 (and its subsections). For samples requiring lysate mixing, follow section 2.2 (and its subsections). The TSP (Total, Soluble, Pellet) analysis that is included in the protocol is useful in optimizing protein expression which can help ensure that approximately equal amounts of α and β subunits are combined during lysate mixing (see Discussion). It also provides an important control if downstream results are not as expected (i.e. it verifies that protein was expressed and soluble). Thus, we highly recommend including TSP analysis initially. Once an optimal protocol has been achieved for a particular subunit combination, TSP analysis is not strictly required which is why it is described as optional below.

  1. Lysis of bacterial cells and preparation of soluble fractions.
    1. Thaw the induced cell pellet on ice for 5 min. Resuspend the pellet in 600 µL of lysis buffer.
    2. Incubate the suspension at 30 ºC with shaking (150 rpm) for 30 min to generate total crude lysate.
      Note: The following step and the subsection that follows are optional.
    3. Remove two 25 μL aliquots from the total crude lysate into separate 1.5 mL microcentrifuge tubes and carry out TSP analysis as follows.
      1. To one of the two 25 μL aliquots, add 5X SDS sample buffer to a final concentration of 1X. Label the tube with "T" for "total crude lysate".
      2. Incubate the "T" sample at 100 ºC for 5 min to completely denature proteins.
      3. Meanwhile, take the second 25 μL aliquot and centrifuge it at 10,000 × g for 10 min. Carefully remove the supernatant without disturbing the tiny pellet, and transfer it to a new 1.5 mL microcentrifuge tube. Label this new tube "S" for "soluble fraction".
      4. To the tiny pellet left behind in the previous step, add 25 μL of lysis buffer and vortex to resuspend. Label this tube "P" for "pellet fraction".
      5. Add 6 μL of 5X SDS sample buffer to the "S" and "P" tubes and incubate at 100 ºC as described above.
        Note: If the TSP samples will not be used that day to analyze expression, they may be frozen at -20 ºC until needed. Once thawed, they must be reincubated at 100 ºC, as described above, immediately prior to loading on a 12% and/or 15% standard SDS-PAGE gel.
    4. While the TSP analysis is carried out, centrifuge the remaining 550 μL of total crude lysate (or the entire 600 μL of total crude lysate if TSP analysis is not carried out) at 10,000 × g for 10 min. Collect the supernatant in a fresh 1.5 mL microcentrifuge tube. This is the soluble lysate that will be used for purification.
  2. Lysate mixing.
    1. Carry out lysis as described in steps 2.1.1 and 2.1.2 above.
    2. Mix 600 µL of total crude lysate from bacteria expressing the desired α subunit with 600 µL of total crude lysate from bacteria expressing the desired β subunit. Incubate at 37 ºC with slow shaking for 30 min.
      Note: It may be necessary to optimize incubation time to achieve maximum assembly during lysate mixing (see Discussion). The time and temperature presented here are optimal for the recombinant proteins in this study and were determined elsewhere18.
    3. Centrifuge the mixed lysate at 10,000 × g for 10 min to separate soluble material from insoluble pellet. Transfer the supernatant to a new 1.5 mL microcentrifuge tube. Use this mixed soluble lysate for protein purification.

3. Protein Purification via Immobilized Cobalt Affinity Resin (ICAR).

  1. Equilibrate the resin.
    1. Thoroughly resuspend the resin by inverting the bottle and transfer 50 µL of the slurry to a 1.5 mL microcentrifuge tube. Centrifuge at 700 × g for 2 min to pellet resin. Carefully aspirate supernatant.
      Note: We carry out pipette aspiration using a blue 1 mL pipette tip to remove the bulk of the liquid. A white 2 μL tip is used for fine control of removal of the remaining supernatant, leaving a very thin layer of liquid covering the beads.
    2. Add 1 mL of Buffer A. Mix gently to resuspend resin and centrifuge at 700 × g for 2 min to pellet resin. Carefully aspirate the supernatant and repeat this wash step one more time.
  2. Apply soluble lysate obtained previously in section 2.1 or 2.2 to the equilibrated resin. Incubate the tube with gentle rotation at 4 ºC for 60 min. Centrifuge the tube at 700 × g for 5 min and carefully aspirate the supernatant.
  3. Wash the resin as follows to remove nonspecifically bound proteins.
    1. Resuspend resin with 1 mL of Buffer A and incubate with gentle rocking at 4 ºC for 10 min. Centrifuge the tube at 700 × g for 5 min and carefully aspirate the supernatant. Repeat this wash step one more time.
    2. Resuspend resin with 1 mL of Buffer B (Buffer A with 5 mM Imidazole) and incubate with gentle rocking at 4 ºC for 5 min. Centrifuge the tube at 700 × g for 5 min and carefully aspirate the supernatant. Repeat this wash step one more time.
    3. Resuspend resin with 1 mL of Buffer C (Buffer A with 10 mM Imidazole) and incubate with gentle rocking at 4 ºC for 5 min. Centrifuge the tube at 700 × g for 5 min and carefully aspirate the supernatant.
  4. Elute the protein by adding 400 µL of Buffer E (Buffer A with 200 mM Imidazole) to the resin. Incubate with gentle rocking at 4 ºC for 5 min. Centrifuge at 700 × g for 5 min.
  5. Transfer supernatant containing purified protein to a new 1.5 mL centrifuge tube.
  6. Desalt the purified protein by serial centrifugation as follows.
    1. To 400 μL of purified protein, add 100 μL Buffer A (this reduces Imidazole concentration from 200 mM to 160 mM). Apply purified protein to 0.5 mL ultracentrifugal filters with a 10 kDa molecular weight cut-off. Centrifuge at 14,000 × g for 5 min.
    2. Discard the filtrate and add 400 μL Buffer A to dilute the retentate and centrifuge again. Continue the cycles of centrifugation/dilution until imidazole concentration falls below 4 mM. As an example, if each centrifugation concentrates 500 μl down to ~70 μl (or approximately 7-fold), then two cycles will reduce the starting 160 mM imidazole concentration (7×7 = 49-fold) to approximately 3.3 mM.
    3. Measure the protein concentration of the desalted sample using the BCA assay21.
      Note: Desalting is required to reduce Imidazole levels below the tolerance limit for the BCA assay, as described in the manufacturer's instructions. Our lab prefers the BCA assay because it is sensitive, has a large dynamic range, and exhibits much less protein-to-protein variation. However, other methods to determine protein concentration can be substituted for the BCA assay. The key is to be aware of each method's advantages and limitations.
  7. Add 5X native sample buffer to a final concentration of 1X and proceed to electrophoresis. Alternatively, store samples at -20 ºC for later analysis.

4. Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE).

Caution: Unpolymerized acrylamide is a neurotoxin. Wear appropriate protective gear.

  1. Prepare the nondenaturing PAGE gel as follows.
    1. Prepare the gel cassette for casting using clean glass plates and casting stand. Place gradient maker on top of a small magnetic stirrer and place a tiny stir bar into each chamber.
    2. Using 40% (w/v) acrylamide and 2% (w/v) bisacrylamide stock solutions, prepare 5% and 10% (w/v) acrylamide gel solutions in a native resolving buffer. Ensure that the ratio of total acrylamide to bisacrylamide is 37.5:1. Chill on ice prior to pouring.
      Note: The nondenaturing PAGE system used here is essentially identical to the Laemmli SDS-PAGE system, with SDS omitted22.
    3. Add Ammonium Persulfate (from a 10% (w/v) stock solution prepared in water) to the acrylamide solutions to initiate polymerization. Final concentration of Ammonium Persulfate is 0.1% (w/v).
    4. Pour the acrylamide solutions into the two chambers of the gradient maker. With the outlet tubing inserted between the plates of the gel cassette, activate the magnetic stirrer and open the gradient maker valves. Pour the 5 - 10% nondenaturing gradient gel.
    5. Once poured, overlay the gel with a thin layer of Isopropanol and allow the gel to polymerize for 30 min. During this time, prepare fresh 5% acrylamide gel solution in native resolving buffer. After the gel sets, pour off Isopropanol and rinse the top of the gel with deionized water from a squirt bottle.
    6. To the 5% gel solution prepared above, add Ammonium Persulfate (exactly as described in 4.1.3) and pour on top of the polymerized gel until glass plates are full. Insert gel comb. Allow the overlaid gel to polymerize for an additional 30 min.
    7. Once gel is polymerized, assemble gel cassette into electrophoresis apparatus. Alternatively, store the gel at 4 ºC, wrapped in moistened paper towels and plastic wrap until ready for use.
  2. Once nondenaturing PAGE gel is assembled into electrophoresis apparatus, fill the tank with 1X native running buffer prepared fresh from a 10X native running buffer stock.
  3. Load 10 μg of purified protein, obtained at the end of section 3, into each well using a glass syringe.
  4. Load 2 μL of high molecular weight native protein standard (diluted in 1X native running buffer) into one of the wells.
  5. Run gel at 55 V and 4 ºC until the dye front runs off the gel (approximately 4 - 4.5 h).
  6. In addition to the nondenaturing gel, analyze aliquots of the purified protein by standard SDS-PAGE22.
    1. Mix aliquots (10 μg) of the purified protein samples, obtained at the end of section 3, with 5X SDS-sample buffer to a final concentration of 1X.
    2. Incubate at 100 ºC for 5 min and load onto standard 12% and/or 15% SDS-PAGE gels22.
    3. Run at 80V for 20 min and then at 120 V until the dye front runs off the gel (this is approximately an additional 75 min for a 12% gel, and 120 min for a 15% gel).

5. Visualizing Activity and Protein Staining.

  1. Following electrophoresis, carefully separate glass plates and transfer the nondenaturing gel to a gel tray containing 50 mL of deionized water. Rinse gel with gentle rocking for 5 min. Discard water and repeat this wash step three times.
  2. Perform substrate overlay assay as follows.
    1. Add 1 mL of developing buffer containing the fluorogenic peptide substrate Suc-LLVY-AMC and spread uniformly over the gel. Incubate at 37 ºC for 30 min.
      Note: A glass rod or a gel releaser (small wedge of plastic used to separate glass plates) can be used to spread the liquid over the gel.
    2. Carefully transfer the gel onto the UV transilluminator of the gel imaging system and observe fluorescence due to the cleaved peptide substrate. Record image. Carefully transfer the gel back to the gel tray.
    3. Rinse the gel twice with 50 mL of deionized water for 5 min with gentle rocking.
    4. Stain the gel with 10 mL of a colloidal coomassie stain reagent for 60 min with gentle rocking. Destain gel with 50 mL water until background becomes clear. This staining step applies to the standard SDS-PAGE gels as well.

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Results

Proteasome assembly (Figure 1) begins when α subunits combine to form rings9. This can be illustrated when α subunits from the archaeon Methanococcus maripaludis S2 are expressed in E. coli as C-terminally hexahistidine tagged (his-tagged) derivatives (Table 1). When the recombinant α-his protein was purified by ICAR and analyzed by nondenaturing PAGE, two bands were observed (Figure 2A, lane 1). We have previously demonst...

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Discussion

We demonstrate the benefit of a combined approach to analyzing proteasome assembly by nondenaturing PAGE using recombinant archaeal proteasomes. The usual method9,11 of bacterial coexpression of proteasome subunits allows for rapid analysis but may not reveal key assembly intermediates. We suggest combining coexpression with lysate mixing to develop a broader picture of assembly events.

The advantage of this combined approach is that despite requiring separate expression of the ^...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported in part by a Research Support Funds Grant (RSFG) from Indiana University-Purdue University, Indianapolis, and in part by an award from the American Heart Association 14GRNT20390154, to A.R.K.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acrylamide (40%) solutionBiorad1610104Unpolymerized acrylamide is a neurotoxin. Wear proper protective gear
Amicon ultra 0.5 mL centrifugal filtersEMDMilliporeUFC501024
Ammonium PersulfateSigmaA3678
ATPSigmaA7699
BCA assay kitPierce23225
Bisacrylamide (2%) solutionBiorad1610142
Bromophenol blueSigmaB8026
DNaseISigmaDN25
Dithiothreitol (DTT)Thermo FisherBP172
E. coli BL21 competent cellsEMD Millipore69450
GelCode BlueThermo Fisher24592Colloidal coomassie stain reagent for gels
Gel doc EZ systemBiorad1708270Gel documentation system
Gel releasersBiorad1653320Wedge shaped plastic used to separate gel plates; useful for spreading liquid.
Glass rodThermo Fisher11-380B
GlycerolSigma49767
GlycineThermo FisherBP3865
Hamilton syringeThermo Fisher14-813-38Glass syringe for loading gels
HEPESUS BiologicalsH2010
HMW Native calibration kitGE Healthcare170445-01High molecular weight protein standards
Hoefer SG30Thermo Fisher03-500-277Gradient maker
ImidazoleUS Biologicals280671
IPTGUS BiologicalsI8500For induction of protein expression
IsopropanolThermo FisherBP26181
Kanamycin sulfateUS BiologicalsK0010
LysozymeSigmaL6876
MgCl2Fluka analytical630680
Mini Protean Tetra CellBiorad1658002EDUGel electrophoresis apparatus
NaClThermo FisherS640-3
NaOHThermo FisherS318-1
Pefabloc SCRoche11429876001Protease inhibitor
pET42EMD Millipore70562Expression plasmid
Precision plus all blue standardBiorad1610373Molecular protein standard for SDS-PAGE
Quickchange mutagenesis kitAgilent technologies200521
Sodium Dodecyl Sulfate (SDS)Thermo FisherBP166
Suc-LLVY-AMCEnzo lifesciencesBML P802-0005Fluorogenic substrate
Talon Metal Affinity ResinClontech635502Immobilized Cobalt Affinity Resin
TEMEDSigmaT7024
TrisUS BiologicalsT8600
Triton-X100Sigma93426
TryptoneBacto BD211699
UV sample trayBiorad1708271For UV imaging of gels
Yeast extractBacto BD212720

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Coexpression ApproachLysate MixingHalf Proteasome DetectionArchaeal 20S ProteasomeAlpha Beta SubunitsBacterial ExpressionProtein Complex Analysis

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