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.

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).
<ol>
	<li>Transform expression plasmid of interest into chemically competent E.coli BL21(DE3) cells.</li>
	<li>Add 1 - 2 &#956;L&#160;of plasmid (typically 50 - 100 ng) to an aliquot of DE3&#160;cells that were freshly thawed on ice, and continue to incubate on ice for 20 min.</li>
	<li>Heat shock the cells at 42 &#176;C for 45 s and return tube to ice for additionl 2 min.</li>
	<li>Add 1 mL of LB medium and incubate at 37 &#176;C with shaking (150 rpm) for 1 h.</li>
	<li>Spread 100 - 200 &#956;L&#160;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 &#186;C O/N.</li>
	<li>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 &#186;C with shaking (150 rpm) for approximately 2.5 - 3 h, or till turbidity is observed.</li>
	<li>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 &#186;C for 40 min.</li>
	<li>Induce protein expression in the liquid cultures by adding IPTG from a stock solution to a final concentration of 1 mM. Incubate at 37 &#186;C with shaking (150 rpm) for approximately 6 - 7 h.</li>
	<li>Harvest bacterial cell cultures in their entirety into 1.5 mL&#160;microcentrifuge tubes.</li>
	<li>Add 1.5 mL&#160;of a culture into microcentrifuge tube. Centrifuge at 10,000 &#215; g for 1 min.</li>
	<li>Discard the supernatant and add another 1.5 mL&#160;of the same culture to the pellet. Centrifuge at 10,000 &#215; g for 1 min.</li>
	<li>Repeat step 1.11 until the entire culture is harvested into the microcentrifuge tube.</li>
	<li>Store the induced pellets at -80 &#176;C until lysis.</li>
</ol>
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 &#945; and &#946; 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.
<ol>
	<li>Lysis of bacterial cells and preparation of soluble fractions.
	<ol>
		<li>Thaw the induced cell pellet on ice for 5 min. Resuspend the pellet in 600 &#181;L&#160;of lysis buffer.</li>
		<li>Incubate the suspension at 30 &#186;C with shaking (150 rpm) for 30 min to generate total crude lysate. 
		Note: The following step and the subsection that follows are optional.</li>
		<li>Remove two 25 &#956;L&#160;aliquots from the total crude lysate into separate 1.5 mL&#160;microcentrifuge tubes and carry out TSP analysis as follows.
		<ol>
			<li>To one of the two 25 &#956;L&#160;aliquots, add 5X SDS&#160;sample buffer to a final concentration of 1X. Label the tube with &#34;T&#34; for &#34;total crude lysate&#34;.</li>
			<li>Incubate the &#34;T&#34; sample at 100 &#186;C for 5 min to completely denature proteins.</li>
			<li>Meanwhile, take the second 25 &#956;L&#160;aliquot and centrifuge it at 10,000 &#215; g for 10 min. Carefully remove the supernatant without disturbing the tiny pellet, and transfer it to a new 1.5 mL&#160;microcentrifuge tube. Label this new tube &#34;S&#34; for &#34;soluble fraction&#34;.</li>
			<li>To the tiny pellet left behind in the previous step, add 25 &#956;L&#160;of lysis buffer and vortex to resuspend. Label this tube &#34;P&#34; for &#34;pellet fraction&#34;.</li>
			<li>Add 6 &#956;L&#160;of 5X SDS&#160;sample buffer to the &#34;S&#34; and &#34;P&#34; tubes and incubate at 100 &#186;C as described above. 
			Note: If the TSP samples will not be used that day to analyze expression, they may be frozen at -20 &#186;C until needed. Once thawed, they must be reincubated at 100 &#186;C, as described above, immediately prior to loading on a 12% and/or 15% standard SDS-PAGE gel.</li>
		</ol>
		</li>
		<li>While the TSP analysis is carried out, centrifuge the remaining 550 &#956;L&#160;of total crude lysate (or the entire 600 &#956;L&#160;of total crude lysate if TSP analysis is not carried out) at 10,000 &#215; g for 10 min. Collect the supernatant in a fresh 1.5 mL&#160;microcentrifuge tube. This is the soluble lysate that will be used for purification.</li>
	</ol>
	</li>
	<li>Lysate mixing.
	<ol>
		<li>Carry out lysis as described in steps 2.1.1 and 2.1.2 above.</li>
		<li>Mix 600 &#181;L&#160;of total crude lysate from bacteria expressing the desired &#945; subunit with 600 &#181;L&#160;of total crude lysate from bacteria expressing the desired &#946; subunit. Incubate at 37 &#186;C 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.</li>
		<li>Centrifuge the mixed lysate at 10,000 &#215; g for 10 min to separate soluble material from insoluble pellet. Transfer the supernatant to a new 1.5 mL&#160;microcentrifuge tube. Use this mixed soluble lysate for protein purification.</li>
	</ol>
	</li>
</ol>
3. Protein Purification via Immobilized&#160;Cobalt Affinity Resin (ICAR).
<ol>
	<li>Equilibrate the resin.
	<ol>
		<li>Thoroughly resuspend the resin by inverting the bottle and transfer 50 &#181;L&#160;of the slurry to a 1.5 mL&#160;microcentrifuge tube. Centrifuge at 700 &#215; g for 2 min to pellet resin. Carefully aspirate supernatant. 
		Note: We carry out pipette aspiration using a blue 1 mL&#160;pipette tip to remove the bulk of the liquid. A white 2 &#956;L&#160;tip is used for fine control of removal of the remaining supernatant, leaving a very thin layer of liquid covering the beads.</li>
		<li>Add 1 mL&#160;of Buffer A. Mix gently to resuspend resin and centrifuge at 700 &#215; g for 2 min to pellet resin. Carefully aspirate the supernatant and repeat this wash step one more time.</li>
	</ol>
	</li>
	<li>Apply soluble lysate obtained previously in section 2.1 or 2.2 to the equilibrated resin. Incubate the tube with gentle rotation at 4 &#186;C for 60 min. Centrifuge the tube at 700 &#215; g for 5 min and carefully aspirate the supernatant.</li>
	<li>Wash the resin as follows to remove nonspecifically bound proteins.
	<ol>
		<li>Resuspend resin with 1 mL&#160;of Buffer A and incubate with gentle rocking at 4 &#186;C for 10 min. Centrifuge the tube at 700 &#215; g for 5 min and carefully aspirate the supernatant. Repeat this wash step one more time.</li>
		<li>Resuspend resin with 1 mL&#160;of Buffer B (Buffer A with 5 mM Imidazole) and incubate with gentle rocking at 4 &#186;C for 5 min. Centrifuge the tube at 700 &#215; g for 5 min and carefully aspirate the supernatant. Repeat this wash step one more time.</li>
		<li>Resuspend resin with 1 mL&#160;of Buffer C (Buffer A with 10 mM Imidazole) and incubate with gentle rocking at 4 &#186;C for 5 min. Centrifuge the tube at 700 &#215; g for 5 min and carefully aspirate the supernatant.</li>
	</ol>
	</li>
	<li>Elute the protein by adding 400 &#181;L of Buffer E (Buffer A with 200 mM Imidazole) to the resin. Incubate with gentle rocking at 4 &#186;C for 5 min. Centrifuge at 700 &#215; g for 5 min.</li>
	<li>Transfer supernatant containing purified protein to a new 1.5 mL&#160;centrifuge tube.</li>
	<li>Desalt the purified protein by serial centrifugation as follows.
	<ol>
		<li>To 400 &#956;L&#160;of purified protein, add 100 &#956;L&#160;Buffer A (this reduces Imidazole concentration from 200 mM to 160 mM). Apply purified protein to 0.5 mL&#160;ultracentrifugal filters with a 10 kDa molecular weight cut-off. Centrifuge at 14,000 &#215; g for 5 min.</li>
		<li>Discard the filtrate and add 400 &#956;L&#160;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 &#956;l down to ~70 &#956;l (or approximately 7-fold), then two cycles will reduce the starting 160 mM imidazole concentration (7&#215;7 = 49-fold) to approximately 3.3 mM.</li>
		<li>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&#39;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&#39;s advantages and limitations.</li>
	</ol>
	</li>
	<li>Add 5X native sample buffer to a final concentration of 1X and proceed to electrophoresis. Alternatively, store samples at -20 &#186;C for later analysis.</li>
</ol>
4. Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE).
Caution: Unpolymerized acrylamide is a neurotoxin. Wear appropriate protective gear.
<ol>
	<li>Prepare the nondenaturing PAGE gel as follows.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Once gel is polymerized, assemble gel cassette into electrophoresis apparatus. Alternatively, store the gel at 4 &#186;C, wrapped in moistened paper towels and plastic wrap until ready for use.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Load 10 &#956;g of purified protein, obtained at the end of section 3, into each well using a glass syringe.</li>
	<li>Load 2 &#956;L&#160;of high molecular weight native protein standard (diluted in 1X native running buffer) into one of the wells.</li>
	<li>Run gel at 55 V and 4 &#186;C until the dye front runs off the gel (approximately 4 - 4.5 h).</li>
	<li>In addition to the nondenaturing gel, analyze aliquots of the purified protein by standard SDS-PAGE22.
	<ol>
		<li>Mix aliquots (10 &#956;g) of the purified protein samples, obtained at the end of section 3, with 5X SDS-sample buffer to a final concentration of 1X.</li>
		<li>Incubate at 100 &#186;C for 5 min and load onto standard 12% and/or 15% SDS-PAGE gels22.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
5. Visualizing Activity and Protein Staining.
<ol>
	<li>Following electrophoresis, carefully separate glass plates and transfer the nondenaturing gel to a gel tray containing 50 mL&#160;of deionized water. Rinse gel with gentle rocking for 5 min. Discard water and repeat this wash step three times.</li>
	<li>Perform substrate overlay assay as follows.
	<ol>
		<li>Add 1 mL&#160;of developing buffer containing the fluorogenic peptide substrate Suc-LLVY-AMC and spread uniformly over the gel. Incubate at 37 &#186;C 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.</li>
		<li>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.</li>
		<li>Rinse the gel twice with 50 mL of deionized water for 5 min with gentle rocking.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 &#94...

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&#160;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, &#945; and &#946;5,7,8. In eukaryotes, the two outer rings are each comprised of seven distinct &#945; subunits and the two inner rings are each comprised of seven distinct &#946; subunits; proteolytic activity resides within three of the &#946; subunits. By contrast, the CP rings of archaea and bacteria are usually comprised of only one type of &#945; and one type of &#946; 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, &#945; subunits assemble into &#945; rings first, which serve as a scaffold onto which &#946; subunits assemble. The resulting half-proteasomes (&#945;7&#946;7) dimerize, giving rise to fully assembled CP (&#945;7&#946;7&#946;7&#945;7). During dimerization, the propeptides present on &#946; 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 &#945; and &#946; 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 &#945; and &#946; 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 &#945; and &#946; 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.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:165px;">Acrylamide (40%) solution</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610104</td>
			<td style="width:309px;">Unpolymerized acrylamide is a neurotoxin. Wear proper protective equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Amicon ultra 0.5ml centrifugal filters</td>
			<td style="width:123px;">EMDMillipore</td>
			<td>UFC501024</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Ammonium persulfate</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">ATP</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">A7699</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">BCA assay kit</td>
			<td style="width:123px;">Pierce</td>
			<td style="width:105px;">23225</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Bisacrylamide (2%) solution</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610142</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Bromophenol blue</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">B8026</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">DNaseI</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">DN25</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Dithiothreitol (DTT)</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP172</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">E.coli BL21 competent cells</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:105px;">69450</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">GelCode Blue</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">24592</td>
			<td style="width:309px;">Colloidal coomassie stain reagent for gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Gel doc EZ system</td>
			<td style="width:123px;">Biorad</td>
			<td>1708270</td>
			<td style="width:309px;">Gel documentation system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:165px;">Gel releasers</td>
			<td style="width:123px;">Biorad</td>
			<td>1653320</td>
			<td style="width:309px;">Wedge shaped plastic used to separate gel plates; useful for spreading liquid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glass rod</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td>11-380B</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">49767</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycine</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP3865</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hamilton syringe</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td>14-813-38</td>
			<td style="width:309px;">Glass syringe for loading gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">HEPES</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">H2010</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">HMW Native calibration kit</td>
			<td style="width:123px;">GE Healthcare</td>
			<td style="width:105px;">170445-01</td>
			<td style="width:309px;">High molecular weight protein standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hoefer SG30</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">03-500-277</td>
			<td style="width:309px;">Gradient maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Imidazole</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">280671</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">IPTG</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">I8500</td>
			<td style="width:309px;">For induction of protein expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropanol</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP26181</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Kanamycin sulfate</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">K0010</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Lysozyme</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">L6876</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:165px;">MgCl2</td>
			<td style="width:123px;">Fluka analytical</td>
			<td style="width:105px;">630680</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Mini Protean Tetra Cell</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1658002EDU</td>
			<td style="width:309px;">Gel electrophoresis apparatus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">NaCl</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">S640-3</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">NaOH</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">S318-1</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Pefabloc SC</td>
			<td style="width:123px;">Roche</td>
			<td style="width:105px;">11429876001</td>
			<td style="width:309px;">Protease inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">pET42</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:105px;">70562</td>
			<td style="width:309px;">Expression plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Precision plus all blue standard</td>
			<td style="width:123px;">Biorad</td>
			<td style="width:105px;">1610373</td>
			<td style="width:309px;">Molecular protein standard for SDS-PAGE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Quickchange mutagenesis kit</td>
			<td style="width:123px;">Agilent technologies</td>
			<td style="width:105px;">200521</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:105px;">BP166</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Suc-LLVY-AMC</td>
			<td style="width:123px;">Enzo lifesciences</td>
			<td style="width:105px;">BML P802-0005</td>
			<td style="width:309px;">Fluorogenic substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:165px;">Talon Metal Affinity Resin</td>
			<td style="width:123px;">Clontech</td>
			<td style="width:105px;">635502</td>
			<td style="width:309px;">Immobilized-cobalt affinity resin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">TEMED</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">T7024</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Tris</td>
			<td style="width:123px;">US Biologicals</td>
			<td style="width:105px;">T8600</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Triton-X100</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:105px;">93426</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Tryptone</td>
			<td style="width:123px;">Bacto BD</td>
			<td style="width:105px;">211699</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">UV sample tray</td>
			<td style="width:123px;">Biorad</td>
			<td>1708271</td>
			<td style="width:309px;">For UV imaging of gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Yeast extract</td>
			<td style="width:123px;">Bacto BD</td>
			<td style="width:105px;">212720</td>
			<td style="width:309px;"></td>
		</tr>
	</tbody>
</table>

examining proteasome assembly with recombinant archaeal proteasomes and nondenaturing page:  the case for a combined approach

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&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; 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 &#945; and &#946; 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 &#945;-ring and one complete &#946;-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.

Proteasome assembly (Figure 1) begins when &#945; subunits combine to form rings9. This can be illustrated when &#945; 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 &#945;-his protein was purified by ICAR&#160;and analyzed by nondenaturing PAGE, two bands were observed (Figure 2A, lane 1). We have previously demonst...

Watch this Scientific Journal Video about Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach at JoVE.com

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

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

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

The overall goal of this experiment is to analyze assembly of a multi-protein complex such as the proteasome, using recombinant subunits, and nondenaturing Polyacrylamide Gel Electrophoresis, or PAGE.This method can help answer key questions in the proteasome field such as, what intermediate species are encountered along the assembly pathway of this protein complex.The main advantage of this technique is that it allows rapid screening of assembly by two parallel approaches, allowing detection of on and off pathway intermediates.This procedure begins with bacterial expression as described in the text protocol.To perform bacterial lysis, thaw the induced cell pallet co-expressing the desired combination of proteasome subunits, on ice for five minutes.Once thawed, resuspend the pallet in 600 microliters of Lysis Buffer.Incubate the suspension at 30

examining-proteasome-assembly-with-recombinant-archaeal-proteasomes

Research

JoVE Journal

Biochemistry

6.6K Views.  Indiana University-Purdue University, Indianapolis (IUPUI). The overall goal of this experiment is to analyze assembly of a multi-protein complex such as the proteasome, using recombinant subunits, and nondenaturing Polyacrylamide Gel Electrophoresis, or PAGE.This method can help answer key questions in the proteasome field such as, what intermediate species are encountered along the assembly pathway of this protein complex.The main advantage of this technique is that it allows rapid screening of assembly by two parallel approaches, allowing detection...

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. However, capillary columns are much less robust in comparison to their higher flow countertypes. Because of easy contamination and blocking, they often need replacement. That makes them a markedly expensive part of the total LC-MS analysis cost. In-house packing of UHPLC capillary columns saves a lot of money and allows customization. However, the standard packing procedure in the 100-bar pressure bomb works well only for HPLC columns but is too slow for UHPLC sorbents. Here we provide a description of an optimized FlashPack protocol applied to the same 100-bar pressure bomb setup. The method is based on packing from ultra-high sorbent concentration slurry and is developed for in-house manufacturing of UHPLC capillary columns of unlimited length in reasonable time.

Here we present a protocol for the optimized FlashPack capillary column packing procedure. Application of an optimized protocol to a common 100-bar pressure bomb setup allows 10-times faster packing and manufacturing of long ultra-high performance capillary columns.

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...