This work was supported by a grant from the NIH (5T32GM008275-22) and an American Heart Association predoctoral fellowship (to E.A.S.); a Chemistry-Biology Interface Fellowship from the NIH (2T32GM071339-06A1) (to M.E.D.); and grants from the NIH (1DP2OD002177-01 and NS067354-0110), The Ellison Medical Foundation, and The Bill and Melinda Gates Foundation (to J.S.).

1. Expression of Hsp104
<ol>
<li>The plasmid employed for purification in E. coli, pPROEX-HTb-Hsp104, contains the Hsp104 open-reading frame under the inducible control of the trc promoter26. The plasmid produces Hsp104 with an N-terminal His6-tag that can be removed by TEV protease cleavage. Transform pPROEX-HTb-Hsp104 into codon-optimized E. coli BL21-CodonPlus-RIL cells (Stratagene, Agilent Technologies) using a typical bacterial transformation procedure (e.g. according to manufacturer's instructions). It is important to use a codon-optimized E. coli strain because Hsp104 has an unusual codon bias.</li>
<li>Inoculate a 100mL 2XYT culture (USB. Recipe: 16g/L casein peptone, 10g/L yeast extract, 5g/L NaCl, pH 7.0) supplemented with 100&mu;g/mL ampicillin and 34&mu;g/mL chloramphenicol with fresh transformants and grow overnight at 37&deg;C with shaking at 200rpm.</li>
<li>Inoculate 10mL of the overnight culture into 6 X 1L 2XYT with 100&mu;g/mL ampicillin and 34&mu;g/mL chloramphenicol in 2L flasks. Shake at 250rpm at 37&deg;C until OD600 = 0.4-0.6. Stop shaking and reduce temperature to 15&deg;C. Allow cells to equilibrate unagitated for 30min in the incubator until it reaches 15&deg;C. Once 15&deg;C is reached induce protein expression by adding IPTG to a final concentration of 1mM. Resume shaking at 250rpm overnight (12-16 hours).</li>
</ol>
2. Cell Harvest and Lysis
<ol>
<li> Harvest cells by centrifugation (in a precooled rotor) at 4,000rpm for 20min at 4&deg;C (we use a Sorvall RC 3BP+ centrifuge). Subsequent steps must be performed immediately because Hsp104 activity is diminished when E. coli cells are frozen. Moreover, all subsequent steps should be performed on ice or at 4&deg;C.</li>
<li>Resuspend cell pellets in prechilled (on ice) 10mL lysis buffer (40mM HEPES-KOH pH 7.4, 500mM KCl, 20mM MgCl2, 2.5% (w/v) glycerol, 20mM imidazole, 5&mu;M pepstatin A, complete protease inhibitor cocktail (1 EDTA-free tablet/50mL), and 2mM &beta;-mercaptoethanol).</li>
<li>Lyse cells with a French press (Emulsiflex) homogenizer that uses a heat exchanger immersed in ice water. Before use, ensure that all cell clumps have been solubilized. After equilibrating homogenizer with lysis buffer, two passes through the cylinder with a pressure of 15,000-18,000psi is sufficient for full lysis. If a French press is not available, incubation with lysozyme followed by sonication can be used (see discussion). Save a small sample of lysed cells for SDS-PAGE analysis (1&mu;L) (Lysate in Fig. 2).</li>
<li>Remove cell debris by centrifugation at 16,000rpm for 20min at 4&deg;C (we use a Sorvall RC5C+ centifuge). Retain supernatant for next step and save a small sample (1&mu;L) of supernatant for SDS-PAGE analysis (Ni Load in Fig. 2).</li>
</ol>
3. Hsp104 Purification
<ol>
<li>Mix supernatant from step 2.4 with 12mL (2mL Ni-Sepharose beads per 1L of cells) of 50% slurry of lysis buffer equilibrated Ni-Sepharose beads (GE).</li>
<li>Rotate sample slowly for 3 hours at 4&deg;C such that Ni-Sepharose remains evenly suspended and frothing is minimized. Shorter incubation times are possible, but will decrease yield. At 4&deg;C in the presence of protease inhibitors, little degradation occurs, and a 3 hour Ni-Sepharose incubation time does not decrease activity of the final product. Collect Ni-Sepharose by centrifugation for 2 min at 4&deg;C at 2,000rpm (Eppendorf 5810R centrifuge). Save a small sample of supernatant for SDS-PAGE analysis (1&mu;L) and discard the rest (Ni Flow Through (FT) in Fig. 2).</li>
<li>Wash the retrieved Ni-Sepharose with 25 column volumes of wash buffer (40mM HEPES-KOH pH 7.4, 150mM KCl, 20mM MgCl2, 2.5% (w/v) glycerol, 20mM imidazole, 2mM &beta;-mercaptoethanol), 5 column volumes of wash buffer with 1M KCl to remove contaminants bound electrostatically to Hsp104, and 25 column volumes of wash buffer. Collect Ni-Sepharose after each wash by centrifugation for 2 min at 4&deg;C at 2,000rpm (Eppendorf 5810R centrifuge). After each wash and centrifugation cycle, remove buffer by aspiration.</li>
<li>To elute Hsp104, mix Ni-Sepharose with 1mL elution buffer (40mM HEPES-KOH pH 7.4, 150mM KCl, 20mM MgCl2, 2.5% (w/v) glycerol, 350mM imidazole, 2mM &beta;-mercaptoethanol) per 1mL Ni-Sepharose and rotate at 4&deg;C for 20 minutes. The high imidazole concentration disrupts the Ni bead-His6 interaction. Remove Ni-Sepharose with empty 1.2 mL spin columns (Bio-Rad) by centrifugation for 2 min at 4&deg;C at 2,000rpm (Eppendorf 5810R centrifuge). Save 1&mu;L of eluate for SDS-PAGE analysis (Ni eluate in Fig. 2). For every liter of cells, &tilde;15mg of Hsp104 is obtained at this step.</li>
<li> Buffer exchange eluate into Buffer Q (20mM Tris-HCl pH 8, 0.5mM EDTA, 5mM MgCl2, 50mM NaCl) using MWCO 30,000 Amicon Ultra (Millipore) 15mL centrifugal concentrator units. First, concentrate protein to &tilde;1.5mL then add 14.5mL of Buffer Q to dilute protein 10-fold. Repeat 3 times for buffer exchange. The increased pH and low salt ensures that Hsp104 has a high negative charge.</li>
<li>Purify Hsp104 via anion-exchange chromatography using a Buffer Q equilibrated Resource Q 6mL column (GE). Before injection, protein is filtered through a low protein binding Millex GP PES Membrane 0.22&mu;m syringe filter (Millipore). We employ a flow rate of 1mL/min. Wash away weakly binding proteins with 1 column volume of 20% Buffer Q+ (20mM Tris-HCl pH 8, 0.5mM EDTA, 5mM MgCl2, 1M NaCl). Elute Hsp104 with linear gradient (20%-50% Buffer Q+) over 5 column volumes (Fig. 3). Collect 1ml fractions. Hsp104 and most variants typically elute at &tilde;400mM NaCl (31mS/cm). Save samples of peak fractions (1&mu;L) for SDS-PAGE analysis (Fig. 3, inset).</li>
<li>To remove His6-tag, exchange protein using the Amicon centrifugal concentrator as describe above (see step 3.5) into cleavage buffer (20mM HEPES-KOH pH 7.4, 140mM KCl, and 10mM MgCl2). Use proTEV protease (Promega) or AcTEV protease (Invitrogen) according to manufacturer's instructions. For Hsp104, we have found that higher ratios of TEV protease: Hsp104 are required for full cleavage. We use a ratio of 1 protease unit per 12&mu;g Hsp104. Cleavage should be performed at 30&deg;C for 2-4 hours, followed by a 16 hour incubation at 4&deg;C. Final Hsp104 concentration should be between 20-75&mu;M monomer. Deplete any remaining His6-tagged Hsp104 and TEV protease with Ni-Sepharose (GE) by adding Ni-Sepharose in excess to the amount of Hsp104 in the cleavage reaction (assume beads can bind 15mg of protein per mL of packed resin). Remove beads with empty spin columns (Bio-Rad) by centrifuging for 2min at 2,000rpm at 4&deg;C. Collect samples before and after cleavage (1&mu;L) for SDS-PAGE analysis to ensure cleavage, and the removal of uncleaved protein (Fig. 4).</li>
<li>Exchange into size exclusion buffer (20mM HEPES-KOH pH 7.4, 140mM KCl, 10mM MgCl2, 0.1mM EDTA, 1mM DTT) using MWCO 30,000 Amicon Ultra (Millipore) as described in step 3.5.</li>
<li>Further purify Hsp104 via size-exclusion chromatography. Use size exclusion buffer equilibrated Superose 6 or Superdex 200 column (both from GE) (Fig. 5). Before injection, protein is filtered through a low protein binding Millex GP PES Membrane 0.22&mu;m syringe filter (Millipore). For less than 10mg of protein, the 10/300 size columns (24mL) can be used and 0.5ml fractions are collected. For samples greater than 10mg, the 12/60 size column (120mL) should be used and 1 ml fractions are collected. Refer to manufacturer's instructions for flow-rates and sample volumes to be loaded. After purification, Hsp104 fractions are pooled as shown in Fig. 5 and concentrated in Amicon Centrifugal Concentrator Units (Millipore) [Editor's Note: Video voice over incorrectly states 0.22&mu;M (micromolar) syringe filter, when it should state 0.22&mu;m (micrometer) syringe filter at timecode 04:38.]. Hsp104 is stored as described below. Due to loss of material in separating Hsp104 degradation products and contaminants the final yield of purified full-length Hsp104 is &tilde;1-3mg per liter of starting culture.</li>
</ol>
4. Hsp104 Disaggregase Activity
<ol>
<li>After purification, it is good practice to assess Hsp104 activity in a disaggregation assay. Typically, we employ a luciferase reactivation assay2 (Fig. 6). In this assay, Hsp104 in conjunction with the Hsp70 chaperone system (Hsp70 and Hsp40) disaggregates urea-denatured firefly luciferase aggregates2. Soluble luciferase catalyzes the oxidation of luciferin to oxyluciferin, a reaction that releases light. By monitoring luminescence, the relative reactivation, and therefore disaggregation, of luciferase can be determined. Hsp104 must be able to synergistically collaborate with the Hsp70 co-chaperone system. The amount of recovered luminescence depends on the exact Hsp70:Hsp40 pair utilized. We routinely employ Hsp72 and Hsp40 (Assay Designs). At a minimum, active Hsp104 should produce a 5-fold increase in reactivation of luciferase when compared to reactivation by Hsp72:Hsp40 alone (Fig. 6). Hsp104 preparations that fail to reach this level of activity are discarded.</li>
</ol>
5. Hsp104 Storage
<ol>
<li>For short-term storage, Hsp104 can be kept at 4&deg;C in size-exclusion buffer. However, activity will decline after 2-3 days at 4&deg;C and sharply after 1 week at 4&deg;C. For disaggregation assays we recommend that Hsp104 should be used as soon as possible after purification. Ideally, Hsp104 should be used immediately for amyloid disaggregation assays.</li>
<li>If protein must be stored long-term, Hsp104 is exchanged into storage buffer (20mM HEPES-KOH pH 7.4, 140mM KCl, 10mM MgCl2, 0.1mM EDTA, 1mM DTT, 10% glycerol (w/v)). 100&mu;l aliquots are snap frozen in liquid nitrogen and stored at -80&deg;C. Freeze-thaw cycles drastically reduce Hsp104 activity and should be avoided. We recommend slowly thawing Hsp104 on ice. Amyloid-disaggregase activity will decline sharply after 1 month at -80&deg;C.</li>
</ol>
6. Representative Results and Figures: 
<img src="/files/ftp_upload/3190/3190fig1.jpg" alt="Figure 1"/> Figure 1. Hsp104 is a Bifunctional disaggregase. Disaggregation of disordered aggregates (shown on left) requires the cooperation of the Hsp70 chaperone system (Hsp70 and Hsp40)2. Hsp104 remodels ordered amyloid aggregates (shown on right) without the aid of Hsp70 and Hsp40 in vitro, but Hsp70 and Hsp40 can improve Hsp104 activity against amyloid26,28. For both types of aggregated structures, Hsp104 couples ATP hydrolysis to substrate translocation through its central channel to promote disaggregation. Tyrosine-bearing pore loops engage and shuttle substrate through the central channel49-52.
<img src="/files/ftp_upload/3190/3190fig2.jpg" alt="Figure 2"/> Figure 2. SDS-PAGE analysis of Ni-sepharose affinity purification step. Lysate, Ni Load, Ni FT and Ni eluate samples were fractionated by SDS-PAGE using a 4-20% Tris-HCl 1.0mm Criterion gel (Bio-Rad) and Coomassie stained. Note that not all the Hsp104 is able to bind to the Ni-sepharose. Broad Range molecular weight markers (Bio-Rad) are shown (left lane).
<img src="/files/ftp_upload/3190/3190fig3.jpg" alt="Figure 3"/> Figure 3. Resource Q purification of Ni-sepharose eluate. Blue trace represents absorbance at 280nm and green trace represents % Buffer Q+ (maximum at 50%). The first peak, which elutes at 20% Q+ contains impurities, degradation products and improperly folded Hsp104. The second and major peak contains properly folded and active Hsp104. Flow rate was 1ml/min. Gradient from 20-50% Q+ is 30 minutes or 5 column volumes. Inset: peak fractions are resolved by SDS-PAGE analysis using a 4-20% Tris-HCl 1.0mm Criterion gel (Bio-Rad) and Coomassie stained. Broad Range molecular weight markers (Bio-Rad) are shown (left).
<img src="/files/ftp_upload/3190/3190fig4.jpg" alt="Figure 4"/> Figure 4. SDS-PAGE analysis of proTEV protease cleavage step. His6-Hsp104 from Resource Q purification was treated with proTEV protease for 4 hours at 30&deg;C and then 16 hours at 4&deg;C. Samples were the fractionated by SDS-PAGE using a 4-20% Tris-HCl 1.0mm Criterion gel (Bio-Rad) and Coomassie stained. Note that proTEV cleaved Hsp104 migrates more rapidly. TEV protease and uncleaved Hsp104 have been depleted with Ni-Sepharose as described in Step 3.7. Broad Range molecular weight markers (Bio-Rad) are shown (left).
<img src="/files/ftp_upload/3190/3190fig5.jpg" alt="Figure 5"/> Figure 5. Size-exclusion purification of Hsp104. Cleaved Hsp104 was further purified via a Superose 6 gel filtration column (10/300, GE). Flow rate = 0.4ml/min. The peak between the dashed lines represents pooled fractions. Inset: peak fractions are resolved by SDS-PAGE analysis using a 4-20% Tris-HCl 1.0mm Criterion gel (Bio-Rad) and Coomassie stained. Broad Range molecular weight markers (Bio-Rad) are shown (left).
<img src="/files/ftp_upload/3190/3190fig6.jpg" alt="Figure 6"/> Figure 6. Luciferase reactivation assay. Denatured firefly luciferase aggregates (50nM) were incubated with either Hsp72 and Hsp40 (1&mu;M) (both from Assay Designs), Hsp104 (6&mu;M monomer) or Hsp104, Hsp72 and Hsp40 for 90min at 25&deg;C. Denatured luciferase is only fully reactivated in the presence of both Hsp104 and Hsp40/Hsp72. Recovered luminescence is measured on an Infinite M1000 plate reader (Tecan). Values represent means&plusmn;SEM (n=3).

No conflicts of interest declared.

Timeline: For maximal Hsp104 activity we recommend that the entire purification scheme be completed as rapidly as possible. However, the number of purification steps makes a demanding schedule that may not always be practical. If the purification steps are carried out as quickly as possible, the time from the end of overnight expression through to the 2-4 hours of incubation at 30&deg;C with TEV protease is approximately 9-11 hours. One potential place to pause is following the TEV cleavage step. If absolutely necessary, Hsp104 may be snap frozen after this step as described above (Step 5.2). Size-exclusion chromatography (Step 3.9) is then performed immediately after thawing and the purified Hsp104 must then be used immediately for biochemical assays. Additionally the induction step can also be streamlined to 2 hours at 37&deg;C although this reduces overall yield.
Potential complications: This purification scheme is straight-forward. However, a few critical steps and alternative procedures are discussed below.
Alternative cell lysis: If a French Press is not available, then lysozyme treatment followed by sonication is an effective cell lysis method. Lysis buffer-resuspended cells are incubated with 20mg of hen egg lysozyme (Sigma) per 1L-cell pellet for 30 min on ice to a final concentration of 2mg/ml lysozyme. Cells are then sonicated for two 30 second bursts at level 9 using a MisonixSonicator 3000 with a microtip. The cell suspension is incubated on ice for 1min in between sonication bursts. Lysis has occurred when the suspension changes viscosity and color. Cell debris is then removed as described above (Step 2.4). French press lysis is preferred to sonication because this minimizes Hsp104 degradation and loss of Hsp104 activity.
TEV Protease Cleavage: Curiously, the pPROEX-Htb-Hsp104 His6 tag is particularly resistant to cleavage by TEV protease and requires approximately twice the amount of TEV protease recommended by the manufacturer. Removal of this tag is essential as the pPROEX-Htb His6-tag interferes with Hsp104 disaggregase activity if it is not completely removed. If fully functional His6-tagged Hsp104 is required (e.g. for depletion experiments26) we recommend using the His6-tagged Hsp104 construct described by Schirmer and Lindquist47, where the His6-tag does not affect disaggregase activity26. This protein can be purified using the protocol described above except the TEV cleavage step is omitted.
The protocol we have outlined above is more facile than previous approaches26,47,48. The rapidity and decreased number of steps allows for less perturbation of the protein and therefore yields highly purified and active Hsp104 suitable for structural and mechanistic studies.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent	</th>
			<th>Company	</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>BL21-CodonPlus-RIL Competent Cells</td>
			<td>Stratagene, Agilent Technologies</td>
			<td>230255</td>
		</tr>
		<tr>
			<td>2XYT broth</td>
			<td>USB	</td>
			<td>75864</td>
		</tr>
		<tr>
			<td>Complete, mini, EDTA-free protease inhibitor tablets</td>
			<td>Roche</td>
			<td>1836170</td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Sigma</td>
			<td>P4265</td>
		</tr>
		<tr>
			<td>Ni-Sepharose 6 Fast Flow</td>
			<td>GE Healthcare</td>
			<td>17-5318-02</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 centrifugal filter units (MWCO 30,000)</td>
			<td>Millipore</td>
			<td>UFC903008</td>
		</tr>
		<tr>
			<td>Resource Q - 6ml column</td>
			<td>GE Healthcare</td>
			<td>17-1179-01</td>
		</tr>
		<tr>
			<td>proTEV Protease</td>
			<td>Promega</td>
			<td>V6052</td>
		</tr>
		<tr>
			<td>AcTEV Protease</td>
			<td>Invitrogen</td>
			<td>12575015</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL</td>
			<td>GE Healthcare	</td>
			<td>17-5172-01</td>
		</tr>
		<tr>
			<td>Hsp40</td>
			<td>Assay Designs</td>
			<td>SPP-400</td>
		</tr>
		<tr>
			<td>Hsp72</td>
			<td>Assay Designs</td>
			<td>ADI-NSP-555</td>
		</tr>		
	</tbody>
</table>

purification of hsp104, a protein disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

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17.3K Views.  University of Pennsylvania. Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Video: Purification of Hsp104, a Protein Disaggregase

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...