Name: generation, purification, and characterization of cell-invasive disc1 protein species
Uploaded: 2012-08-30T12:00:00
Duration: 14 min 33 s
Description: Watch this Scientific Journal Video about Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species at JoVE.com

This work was funded by NEURON-ERANET DISCover (BMBF 01EW1003) to C.K. and J. P. H., DFG (Ko 1679/3-1; GRK1033) to C.K. V.B. is supported by a grant of the Forschungskommission of the Medical Faculty of the University of D&uuml;sseldorf.

1. Preparation of Aggresome Donor Cells: Transfection of Monomeric Red Fluorescent Protein (mRFP) or Enhanced Green Fluorescent Protein (eGFP)-tagged Human Full Length (FL) DISC1
<ol>
 <li>Seed 1.5 x 106/ 10 cm dish human neuroblastoma NLF (NLF; Children's Hospital of Philadelphia, Philadelphia, PA) cells in ten 10 cm dishes and grow overnight. NLF cells are cultured in RPMI-1640 medium supplemented with 10% FBS, Penicillin/Strepomycin, L-Glutamine. The next day, cells should be at 70-80% confluency. </li>
 <li>Transiently transfect each 10 cm dish with 15 &mu;g plasmid DNA and Metafectene transfection reagent (Biontex, Martinsried, Germany). In brief, for a 10 cm dish, 15 &mu;g plasmid DNA and 30 &mu;l Metafectene were added to 750 &mu;l Opti-MEM, mixed and incubated at RT for 20 min. 5 dishes were transfected with pcDNA3.1 mRFP/eGFP-tagged human (FL) DISC1 and 5 dishes with the eGFP-plasmid alone. </li>
</ol>
2. Aggresome Purification from Transfected Donor Cells
The protocol described here is a combination of a method to purify Lewy-Bodies from brain tissue13 and a protocol to isolate large aggregates and aggresomes14. Since already existing methods are based on the usage of detergents, the isolation of detergent-free protein for the application in cell-invasiveness assays is critical.
<ol>
 <li>48 hr after transfection, monitor the expression of eGFP and mRFP/eGFP-DISC1 and confirm the formation of aggresomes under a fluorescence microscope (Figure 1). </li>
 <li>Wash the cells with PBS pH 7.4, scrape with 400 &mu;l PBS each, add 1X Protease Inhibitor cocktail (Roche, Indianapolis, IN) and disrupt the cells using a Precellys24 homogenizer (Bertin technologies, France). Briefly, add 1 ml cell suspension to a 2 ml lysis tube and add 10 ceramic beads. Lyse the cells with 3 x 60 sec pulses. The mechanical force of the process may increase the temperature within the sample above 37 &deg;C so care should be taken to chill the sample on ice after the lysis procedure. </li>
 <li>Cool the cells on ice and add 10 mM MgCl2, 40 U/ml DNase A and incubate for 60 min at 37 &deg;C </li>
 <li>Prepare solutions of 80%, 50%, 20% sucrose (w/v) in PBS pH 7.4 (10 ml each). </li>
 <li>Prepare the sucrose gradient in a 15 ml falcon tube, starting with 2 ml 80% sucrose, followed by 50% and 20%. Pour the gradient slowly and be careful not to disturb already poured layers. Layer the digested lysate on top of the gradient and centrifuge for 15 min at 1000 x g at 4 &deg;C. </li>
 <li>Carefully collect the interphase between the 50-80% sucrose layer with a pipette and mix with 5 volumes of PBS pH 7.4. Centrifuge for 15 min at 1000 x g at 4 &deg;C. Repeat washing two additional times. In this interphase, aggresomes are fluorescent and can be visualized in a microscope under UV light. </li>
 <li>Discard the supernatant and resuspend the pellet in 250-500 &mu;l PBS pH 7.4. This pellet contains the purified aggresomes (Figure 2). </li>
</ol>
3. Treatment of Recipient SH-SY5Y Cells with mRFP/eGFP-DISC1 Aggresomes 
<ol>
 <li>Seed 5 x 104 recipient SH-SY5Y human neuroblastoma cells constitutively expressing GFP-DISC1(598-854) on sterile glass coverslips in each well of a 24 well plate. SH-SY5Y cell are cultured in DMEM/F-12 medium supplemented with Penicillin/Streptomycin, non-essential amino acids (NEAA) and 10% FBS. </li>
 <li>24 hr later, add 10 &mu;l to each well and incubate for 48-72 hr. </li>
 <li>Wash the cells 3 x with PBS pH 7.4 and quench extracellular fluorescence with 0.04% Trypan Blue for 5 min. </li>
 <li>Fix the cells with 4% PFA in PBS pH 7.4 for 10 min on ice, wash once with sterile H2O and mount the coverslips on glass slides with ProLong Gold with DAPI mounting medium (Invitrogen, USA). </li>
 <li>Confirm the uptake/invasion of exogenously applied aggresomes by colocalization with recruited proteins expressed by recipient cells in Z-stack images. </li>
</ol>
Yet, uptake/invasion of exogenous protein might not essentially be detected in parallel with recruitment of host cell protein. In our example mRFP-tagged DISC1 aggresomes recruit soluble GFP-tagged DISC1(598-854) protein expressed by the host cell (see Figure 3). Pictures were taken on a Zeiss LSM 510 confocal- or a Zeiss Axiovision Apotome2 Microscope. 
4. Generation of Recombinant DISC1(598-785)
In a previous publication we analyzed the ability of various DISC1 protein fragments to self-interact based on the presence of self-association domains. Among all protein fragments tested human DISC1(598-785) displayed the strongest multimerization propensity4 (Figure 4). Therefore, human DISC1(598-785) was cloned into pET15b (Novagen, Madison, Wisconsin) containing an N-terminal 6-histidine tag, expressed in E. coli BL21-(lDE3) Rosetta (Novagen, US), and purified under denaturing conditions in 8 mol/l urea as described4. In short, the protocol is outlined below.
<ol>
 <li>BL21-(IDE3) Rosetta E. coli were grown in 2 x 500 ml 2YT containing 5 mM-arginine-HCl, 5mM MGSO4, 100 &mu;g/ml carbencillin, 35 &mu;g/ml chloramphenicol up to an OD600: 0.6-0.8 and DISC1(598-785) expression was induced with 1 mM IPTG. </li>
 <li>DISC1(598-785) was expressed for 4 hr at 37 &deg;C, the expression was terminated by harvesting the bacteria by centrifugation. </li>
 <li>The bacterial pellet was resuspended in 50 ml TE buffer (50 mM TRIS-HCl pH 8.0, 5 mM EDTA) plus 2 mM PMSF, 1% TX-100, 250 &mu;g/ml lysozyme, 20 mM MgCl2 and 400 U/50 ml DNase I. To ensure complete lysis, the reaction was incubated for 30 min at RT with gentle stirring.</li>
 <li>Add 10 mM &beta;-mercaptoethanol (ME) and 500 mM NaCl and incubate for another 30 min. </li>
 <li>Spin down bacterial inclusion bodies at 20.000 g for 30 min at 4 &deg;C. </li>
 <li>Resuspend pellet in 50 ml extraction buffer containing 50mM Tris pH 8.0, 5 mM imidazole, 500 mM NaCl, 8 M urea and 10 mM &beta;-ME and remove debris by centrifugation at 20.000 g for 30 min at 4 &deg;C. </li>
 <li>Wash the Ni-NTA agarose matrix with extraction buffer. Incubate the resuspended, precleared protein extract with Ni-NTA matrix for 2 hr at RT. </li>
 <li>Wash the Ni-NTA matrix with extraction buffer containing 12 mM imidazole. </li>
 <li>Elute the protein slowly with 15 ml elution buffer containing 50 mM Tris, 500 mM NaCl, 300 mM imidazole, 8 M urea, 1 mM PMSF, 5 mM EDTA and 10 mM &beta;-ME. </li>
 <li>Dialyze the protein stepwise to PBS pH 7.4 containing 10 mM &beta;-ME. </li>
</ol>
From 1 L bacterial starting culture it is possible to isolate up to 50 mg of recombinant DISC1(598-785) protein. 
&alpha;-synuclein refolding
For experiments with &alpha;-synuclein, 500 &mu;g of the recombinant protein (Sigma-Aldrich, USA) was dissolved in PBS at a concentration of 1 mg/ml. To obtain oligomers, refolding was performed overnight at 37 &deg;C as described in a previous publication10.
5. Labeling of Recombinant DISC1(598-785) with DyLight594
<ol>
 <li>Prior the subsequent labeling process, DISC1(598-785) protein has the be freed from &beta;-ME. Therefore, the protein is dialyzed 3 times to PBS pH 7.4 at a dilution of 1:2,000. </li>
 <li>Label 1 mg DISC1(598-785) with DyLight 594 maleimide (Thermo Scientific, USA) according to the manufacturers instructions. In short, dissolve 1 mg/ ml protein in PBS pH 7.4 and add 5 mM TCEP to recover free thiol groups. Add 20 &mu;l of the DMF dissolved dye to the reaction and incubate for 2 hr at RT. </li>
 <li>Dialyze the protein 3 x to PBS pH 7.4 at a ratio of 1:2000 for 2 hr each. </li>
</ol>
To further increase the purity of the labeled protein a His6-tagged based affinity-purification on a Ni-NTA column is performed. 
<ol start="4">
 <li>Wash 1 ml Ni-NTA matrix (Qiagen, Germany) with 10 ml PBS pH 7.4. </li>
 <li>Apply the labeled, dialyzed protein to the column and let it run over the column slowly. </li>
 <li>Wash the protein with 3 x 20 ml PBS pH 7.4 </li>
 <li>Elute the protein with PBS pH 7.4, 500 mM imidazole dropwise while monitoring the colored band on the Ni-NTA column to reduce the eluate volume. </li>
 <li>Dialyze the eluate 3 x to 10 mM NaPi pH 7.4 at a dilution of 1:2000 for 2 hr each. </li>
 <li>Use a sterile 0.45 &mu;m syringe filter to filter the eluate, aliquot in 5 x 100 &mu;l samples and snap freeze in liquid nitrogen. The total amount of protein should be 0.5 - 1 &mu;g/ml in each 100 &mu;l aliquot. </li>
</ol>
optional concentration step
<ol start="10">
 <li>For stereotactical rat injections, the protein was concentrated 10-fold in a speed-vac (Eppendorf Concentrator 5301). The final buffer condition was 100 mM NaPi. 
 </li>
</ol>
&alpha;-synuclein Labeling
 The labeling and purification (Ni-NTA column) of the recombinant &alpha;-synuclein was performed in parallel to DISC1(598-785). The total starting material was 500 &mu;g instead of 1 mg for DISC1(598-785). 
6. Treatment of Recipient SH-SY5Y Cells with Labeled Recombinant DISC1 (598-785) Protein
<ol>
 <li>Seed 5x104 recipient SH-SY5Y human neuroblastoma cells constitutively expressing GFP-DISC1(598-854) on sterile glass coverslips in each well of a 24 well plate. </li>
 <li>Add labeled protein to the cell culture medium at a concentration of 5-10 &mu;g/ml and incubate for 48-72 hr. </li>
 <li>Wash the cells 3 x with PBS pH 7.4 and quench extracellular fluorescence with 0.04% Trypan Blue for 5 min. </li>
 <li>Fix the cells with 4% PFA in PBS pH 7.4 for 10 min on ice, wash once with sterile H2O and mount the coverslips on glass slides with ProLong Gold with DAPI mounting medium (Invitrogen, USA). </li>
 <li>Confirm the uptake/invasion of exogenously applied recombinant protein by colocalization with recruited proteins expressed by recipient cells in Z-stack images generated by laser scanning confocal microscopy. Yet, recruitment of endogenous protein might not essentially be detected in parallel with invasion of host cell protein, Z-stack images can still confirm the invasive nature of the protein. </li>
</ol>
In our example rec. DISC1(598-785)* at least in part recruits soluble GFP-tagged DISC1(598-854) protein expressed by the host cell (see Figure 5A). For recombinant &alpha;-synuclein invasion but no recruitment is shown (Figure 5B). Pictures were taken on a Zeiss LSM 510 confocal- or a Zeiss Axiovision Apotome2 Microscope. 
The injection of the concentrated (ca. 4 &mu;l of 2.5 &mu;g/&mu;l), labeled DISC1(598-785)* into the mPFC results in the spreading of the protein around the injection site which can be detected even after perfusion of the animal with 4% PFA in PBS pH 7.4 with a rhodamine filterset. In our example DISC1(598-785) is taken up by a distinct number of neurons and can be monitored with Z-stack imaging (Figure 6). 
In general, the injection of proteins other then the ones used here do not necessarily lead to cell-invasion. The invasion event is essentially dependent on the nature of the protein, nevertheless a clean purification is a prerequisite for further analysis.
7. Representative Results
Aggresomes consisting of recombinant FL DISC1-eGFP purified from NLF cells invaded recipient SH-SY5Y cells at low efficiency (approximately 0.3% 5) as seen by colocalization with confocal microscopy (Figure 3). As previously reported, DISC1(598-785) expressed and purified from E. coli formed multimers 4 were equally cell-invasive at an efficiency of approximately 20% 5 (Figure 5A) similar to that of &alpha;-synuclein that was used in parallel as positive control (Figure 5B). Labeled recombinant DISC1(598-785) expressed and purified from E. coli was also cell-invasive to neurons in vivo when the multimeric protein was stereotactically injected into the medial prefrontal cortex of a rat (Figure 6; Pum, Bader, Huston, Korth, unpublished).
<img src="/files/ftp_upload/4132/4132fig1.jpg" alt="Figure 1" /> 
Figure 1. NLF neuroblastoma cells transiently expressing FP-DISC1 (red). Red fluorescent aggresomes can be detected within the cells.
 <img src="/files/ftp_upload/4132/4132fig2.jpg" alt="Figure 2" /> 
Figure 2. Purified mRFP-tagged DISC1 aggresomes after the purification in a sucrose gradient. 
 <img src="/files/ftp_upload/4132/4132fig3.jpg" alt="Figure 3" /> 
Figure 3. Fluorescent picture of invaded aggresomes. mRFP-tagged flDISC1 aggresomes were purified and incubated with SH-SY5Y neuroblastoma cells overexpressing soluble GFP-tagged DISC1(598-854). An uptake of labeled aggresomes is monitored by Z-stack imaging on a Laser-Scan microscope (Zeiss LSM 510).
<img src="/files/ftp_upload/4132/4132fig4.jpg" alt="Figure 4" /> 
Figure 4. SEC-profile of rec. DISC1(598-785) containing the S704 and the C704 single nucleotide polymorphisms (SNP). Both variants show a disruption of ordered oligomerization and the formation of high molecular weight multimers (red arrow). This picture is modified from the publication of Leliveld et al., Biochemistry 2009.
<img src="/files/ftp_upload/4132/4132fig5.jpg" alt="Figure 5" /> 
Figure 5. Fluorescent Z-stack picture of invasive DyLight-labeled recombinant DISC1(598-785). SH-SY5Y neuroblastoma cells expressing soluble GFP-DISC1(598-854) were incubated with labeled, recombinant protein at a concentration of 5 &mu;g/ml. (A) Red aggregates shows the invasion of rec. DISC1(598-785) protein and yellow dots indicate a recruitment of soluble GFP-DISC1(598-854) into aggregates. (B) Recombinant &alpha;-synuclein (red) invades the cells without recruitment with a frequency of about 20%. <a href="https://www.jove.com/files/ftp_upload/4132/4132fig5large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/4132/4132fig6.jpg" alt="Figure 6" /> 
Figure 6. Immunofluorescent picture of the injected labeled, recombinant DISC1(598-785) protein. Z-stack imaging confirms the presence of rec. DISC1(598-785) protein in rat cortical neurons stained with an antibody against neuronal nuclei (NeuN, green).

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No conflicts of interest declared.

In this study we describe the purification of mRFP/eGFP-DISC1 aggresomes from a transfected neuroblastoma cell line, the preparation and labeling of a recombinant DISC1 protein species and their application in cell-invasion experiments of recipient cells in vitro and in vivo.
The purification of native mRFP/eGFP-DISC1 aggresomes was developed from protocols to isolate Lewy body-like structures and larger aggregates13, 14, but modified to avoid the use of detergents and to minimize the risk of false positive cell-invasion that might occur due to detergent-facilitated membrane penetration. 
One possible future improvement might be the in to further increase the purity of the aggresomes by sonication to completely separate remaining membranes and cytoskeleton from the aggresomes. By increasing overall purity, the number of total invasion events that recorded for large aggresomes may be increased5. Whether the limited definition of our suggested 3-phase sucrose is sufficient for aggresomes of other protein species has to be tested, in this sense the protocol outlined here should be considered as a starting point for individual optimization. The purified aggresomes proved to be quite robust in our hands, additional washing steps (without detergent) to eliminate traces of sucrose did not significantly reduce the overall yield. 
In a previous publication we described that oligomer assembly of DISC1 is dependent on distinct multimerization domains in the C-terminus4 (Figure 4). We chose this fragment for recombinant soluble expression in E. coli and subsequent and Ni-NTA purification. To monitor its multimerization and to compare its cell invasiveness with a described cell-invasive protein like &alpha;-synuclein, we labeled DISC1(598-785) with the fluorescent dye DyLight594, and compared its cell-invasiveness with that of equally labeled &alpha;-synuclein. The cell-invasiveness of the recombinant DISC1 fragment was dramatically increased compared to that of native, full length DISC1 aggresomes, likely due to its smaller size and much higher purity. Again, purity seems to play a decisive role in cell-invasiveness.
In our example the invasive aggresomes recruited soluble homologue protein of the target cell line that was recombinantly expressed in a soluble, i.e. cell-dispersed form. The expression of a fluorescent protein (e.g. GFP or RFP) in the recipient cell line is an advantage since it allows the colocalization of invasive aggresomes and recombinant proteins within the recipient cell limit via Z-stack imaging. 
Cell-invasive DISC1 aggresomes and multimeric fragments expressed and purified from E. coli could be a defining feature of a protein conformational diseases relating to DISC1, DISC1opathies 15.
Trouble shooting:
Low aggresome yield after sucrose gradient purification:
The sucrose gradient introduced in this protocol works well with eGFP/mRFP-DISC1(FL) aggresomes. Aggresomes consisting of other proteins might be of variable dimensions therefore the sucrose concentrations should be optimized by other users.
Insufficient purification of recombinant protein:
Any bacterial contaminants in the purification process of the recombinant protein will also be labeled in later steps of the protocol. To avoid contaminations, run the protein on a SDS-PAGE and confirm that the recombinant protein is at least 95% pure by staining with Coomassie-Blue. To ensure highest quality and yield, the protocol has to be optimized for each individual protein.
Low labeling efficiency of recombinant proteins:
Any contaminations that contain free SH-groups will decrease efficient labeling of the protein. Therefore, extensive dialysis and Ni-NTA based purification is mandatory.

<table border="1">
 <tbody>
<tr>
 <td>Reagent name</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>11875-093</td>
 <td>Medium is dependent on the host cell line. The optimal recipient cell line should be determined by the user.</td>
 </tr>
 <tr>
 <td>DMEM/F-12</td>
 <td>Invitrogen</td>
 <td>11320-033</td>
 <td>Medium is dependent on the recipient cell line. The optimal recipient cell line should be determined by the user.</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Metafectene</td>
 <td>Biontex</td>
 <td>T020-1.0</td>
 <td>Other transfection reagents might work as well but were not tested with our protocol.</td>
 </tr>
 <tr>
 <td>Ni-NTA agarose</td>
 <td>Qiagen</td>
 <td>30210</td>
 <td>The experiments were done with Ni_NTA agarose from Qiagen, other suppliers should work as well.</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td>04716728001</td>
 <td>There is no need for RNase free DNase in the process of aggresomes purification.</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>S0389</td>
 <td></td>
 </tr>
 <tr>
 <td>Opti-MEM</td>
 <td>Invitrogen</td>
 <td>31985-062</td>
 <td>Serum-free medium works as well</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140122</td>
 <td>Supplement for SH-SY5Y and NLF medium</td>
 </tr>
 <tr>
 <td>Non-essential-amino-acids (NEAA)</td>
 <td>Sigma-Aldrich</td>
 <td>M7145</td>
 <td>Supplement for SH-SY5Y medium</td>
 </tr>
 <tr>
 <td>Trypan Blue 0.4%</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td>Toxic reagent</td>
 </tr>
 <tr>
 <td>DyLight 594 Maleimide</td>
 <td>Thermo-Fisher Scientific</td>
 <td>46608</td>
 <td>Reduced cysteine reactive dye to form stable thioether bonds. Also available in other colors.</td>
 </tr>
 <tr>
 <td>ProLong Gold with DAPI</td>
 <td>Invitrogen</td>
 <td>P36935</td>
 <td>This antifade liquid mountant gave superior results in our hands.</td>
 </tr>
 <tr>
 <td>Protease Inhibitor cocktail</td>
 <td>Roche</td>
 <td>11873580001</td>
 <td>Dissolve 1 table in 500 &mu;l H2O for 100X solution.</td>
 </tr>
 <tr>
 <td>Synthetic a-synuclein</td>
 <td>Sigma</td>
 <td>S7820</td>
 <td>Refold as described in text.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table of specific reagents.</td>
 </tr>
 <tr>
 <td>Precellys 24</td>
 <td>Bertin Technologies</td>
 <td>03119.200.RD000 </td>
 <td>We have not tested other mechanical homogenizers other than this. Other detergent free homogenization method might work as well, but have not been tested for this protocol.</td>
 </tr>
 <tr>
 <td>5301 Concentrator (speedvac)</td>
 <td>Eppendorf</td>
 <td>5301 000.210</td>
 <td>Only necessary if protein has to be concentrated.</td>
 </tr>
 <tr>
 <td>LSM 510 and Axiovision Apotome2 </td>
 <td>Zeiss</td>
 <td>See manufacturers catalog</td>
 <td>Both microscopes harbor the ability to perform Z-stack imaging. This is a prerequisite for solid and serious evaluation of invasion events.</td>
 </tr>
 <tr>
 <td>Cell culture plastic material</td>
 <td>Nunc</td>
 <td>10 cm dishes #172958, 24-well plates #142475</td>
 <td>The use of different plastic material might influence the interaction of recombinant proteins or aggresomes and the plastic surfaces. We have not tested materials other than the ones described here.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table of specific material and equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
<tr>
 <td>Number</td>
 <td>Buffer name</td>
 <td>content</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1.</td>
 <td>Bacterial growth medium</td>
 <td>16 g/l Bacto Tryptone, 10 g/l Bacto Yeast extract, 5 g/l NaCl, 5 mM-arginine-HCl, 5mM MGSO4, 100 &mu;g/ml carbencillin, 35 &mu;g/ml chloramphenicol</td>
 <td>Grow bacteria to OD600: 0.6-0.8 induce expression with 1 mM IPTG.</td>
 </tr>
 <tr>
 <td>2.</td>
 <td>Bacterial resuspension buffer</td>
 <td>50 mM TRIS-HCl pH 8.0, 5 mM EDTA, 2 mM PMSF, 1% TX-100, 250 &mu;g/ml lysozyme, 20 mM MgCl2 and 400 U/50 ml DNase I</td>
 <td>Resupend bacterial pellet from 1 L overnight culture in 50 ml resuspension buffer.</td>
 </tr>
 <tr>
 <td>3.</td>
 <td>Protein extraction buffer</td>
 <td>50 mM Tris pH 8.0, 5 mM imidazole, 500 mM NaCl, 8 M urea and 10 mM b-ME</td>
 <td></td>
 </tr>
 <tr>
 <td>4.</td>
 <td>Ni-NTA column wash buffer</td>
 <td>50 mM Tris pH 8.0, 5 mM imidazole, 500 mM NaCl, 8 M urea and 10 mM b-ME, 12 mM imidazole</td>
 <td>Modified extraction buffer with 12 mM imidazole added.</td>
 </tr>
 <tr>
 <td>5.</td>
 <td>Ni-NTA column elution buffer</td>
 <td>50 mM Tris, 500 mM NaCl, 300 mM imidazole, 8 M urea, 1 mM PMSF, 5 mM EDTA and 10 mM b -ME </td>
 <td>The elution buffer contains 10 mM b -ME, which is dialyzed later on. </td>
 </tr>
 <tr>
 <td>6.</td>
 <td>PBS</td>
 <td>8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na2HPO4, 0.24 g/l KH2PO4, adjust to pH 7.4</td>
 <td></td>
 </tr>
 <tr>
 <td>7.</td>
 <td>Labeling buffer</td>
 <td>PBS containing 5 mM TCEP</td>
 <td></td>
 </tr>
 <tr>
 <td colspan="4">Table of recipes.</td>
 </tr>
</tbody>
</table>
</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species at JoVE.com

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

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 ...

generation-purification-characterization-cell-invasive-disc1-protein

Research

JoVE Journal

Biology

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 ...

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.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

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 ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

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.

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 ...

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 ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

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 ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...