We acknowledge Dr. Amir Aharoni for his support and Geula Davidov, Noam Grimberg and Chen Guttman for their advice and comments.

1. Cloning and Expression of mamA Gene in E. coli
The mutant gene mamA&Delta;41 was amplified using the polymerase chain reaction (PCR) from genomic DNA of Magnetospirillum magneticum AMB-1, with primers: 5'-GCATTACGCATATGGACGACATCCGCCAGGTG-3' and 5'-GCGCGGCAGCCATA-TGGCATACG-3'. In the amplified DNA fragments, an NcoI site was introduced at the initiation codon ATG and the termination codon was replaced with a ScoI site. The fragments were digested with NcoI and SacI and cloned into the respective sites of pET52b(+), giving rise to pET52bMamA&Delta;41-AMB1. In this construct, the mamA&Delta;41 gene was fused in-frame with the 10-His tag at the C-terminus. The plasmid was electroporated into E. coli strain BL21. E. coli strain BL21 harboring pET52bMamA&Delta;41 was grown in auto-induction (15) medium containing ampicillin (50 mg/ml) 310&deg;K for 3 hours. The cultivation temperature was then shifted from 310&deg; to 300&deg;K and maintained for an additional 48 h at 300&deg;K. The cells were harvested by centrifugation at 5465g for 10 min at 277&deg;K. 8 liters culture produced 60 grams of wet cell pellet.
2. Bioinformatics Calculations
Calculations of molecular weight (MW), according to amino acids sequence and the predicted absorption of 1mg/1ml of protein in 280nm, using the ProtParam server (http://www.expasy.ch/tools/protparam.html). For 10His-Tag-MamA&Delta;41 the MW is 22529 Da (240 amino acids) and for MamA&Delta;41 (with 9 amino acids left after His-tag removal by Thrombin) the Mw is 20596.5Da (187 amino acids). The predicted absorption of 1mg/1ml of protein in 280nm for 10His-Tag-MamA&Delta;41 is 0.595 and for MamA&Delta;41 is 0.579. In addition, the amino acid sequence does not contain any cysteine residues. Therefore, reducing agents are not required during the purification process.
3. Purification of MamA&Delta;41
<ol>
 <li>For stock solution preparation, weigh out and prepare (200 ml each) in the following concentrations: Imidazole (MW 228.2 g/mol) 4M solution, NaCl (Mw 58.44 g/mol) 5M solution, Tris-HCl (Mw 121.3 g/mol) 1M solution, adjust pH=8. Add double distilled water (DDW) and mix using a vortex until the solution is clear. Filter solutions with 0.22&mu;m filter and keep at room temperature.</li>
 <li>Prepare and filter 6 fresh buffers from stock solutions (Table1). </li>
 <li>Dilute and suspend cells in buffer A in 1:2 ratio, grams of cells to buffer volume (ml).</li>
 <li>Add 10&mu;l for each 10gr of bacteria of DNase I (1 mg/ml) in order to break DNA fragments in the sample. Add 1ml for each 20gr of bacteria of EDTA free protease inhibitor cocktail. Incubate for 20 min on ice.</li>
 <li>Disrupted cells by two cycles of French press at 25,000 psi. Unlike sonication, the French press is aimed at high volume (&gt;10ml) cell extrusion by forcing the cells under high pressure through a thin orifice. The piston should be cooled on ice for ten minutes prior to its usage and assembly due to temperature buildup.</li>
 <li>Transfer the lysed cells into 25ml ultracentrifuge tubes and balance them precisely ( 50mg) with buffer A until the sample reaches the tube s neck line.</li>
 <li>Separate cell debris by Ultra centrifugation at 270,000g for 1 hour at 277&deg;K.</li>
 <li>Prepare a homemade gravity Ni NTA column (2.5cm diameter) by adding 4 ml of Ni NTA resin, which are preserves in 20% ethanol, onto the column. Let the fluid drop completely, then wash with 40 ml of DDW and let the fluid drop completely. Pre-equilibrated the resin in the column with 40 ml of buffer A.</li>
 <li>Apply the soluble fraction from ultra-centrifugation tubes on gravity Ni NTA column. Let the fluid drop, collect flow-through (unbound proteins) and preserve at 277K.</li>
 <li>Collect pellet sample from the ultra-centrifugation tubes by using a small tip to scrap the pellet. Mix the tip in 50&mu;l of 5% &beta;-mercaptoethanol in 2X SDS-PAGE sample buffer.</li>
 <li>Wash the column with 100 ml buffer B. Let the fluid drop, collect flow-through and preserve at 277K.</li>
 <li>Prepare 15 marked eppendorf tubes (1.5ml). </li>
 <li>Elution - close column valve and then load 1ml of buffer C onto the column; incubate for 3 min followed by valve opening and flow-through collection. Repeat this step three times.</li>
 <li>Elute rest of suspension at 1 ml buffer C increments and close the valve.</li>
 <li>Measure OD at 280nm of the fractions and evaluate location of the protein peak; if the OD is still &gt;0.3, additional samples are needed.</li>
 <li>SDS-PAGE analysis of collected fractions. Such analysis should be performed also with the following samples, unbound proteins flow-through, wash flow-through and ultra-centrifugation pellet sample. Mix 10&mu;l from each sample with 10&mu;l of 5% &beta;-mercaptoethanol in 2X SDS-PAGE sample buffer. Load the samples on 15% SDS-PAGE and run for 45 min in 180 Volts.</li>
 <li>Stain the gel in 15 ml of InstantBlue solution, shake for 1 hour. Make sure that the plastic box is covered and protected from light.</li>
 <li>Evaluate gel; if protein expression is high and specific according to the indicative bands, merge selected eluted fractions.</li>
 <li>In order to remove 10-His-tag, add bovine thrombin (10 units/&mu;l), 10&mu;l thrombin per 1 mg protein, according to the manufacturer's recommendation to the combined sample.</li>
 <li>Dialysis is used for excess Imidazole removal and to decrease NaCl concentrations for levels needed during ion exchange binding. For dialysis, cut desired length of 7 kD molecular weight cut off (MWCO) dialysis tubing. Wash with DDW and make a knot at one end (or seal with clips). Transfer merged protein solution onto the bag while leaving some space at top and clamp up. </li>
 <li>Submerge the dialysis tubing in 4 Liters chilled dialysis buffer (Buffer D). Stir the solution slowly over night at 277&deg;K.</li>
 <li>Take the dialysis tubing out of the beaker, make a small cut and transfer the protein to 50ml tube.</li>
 <li>Ion-exchange chromatography: assemble pre-packed MonoQ 4.6/100 PE column on Fast Performance Liquid Chromatography (FPLC). Wash the column with 5 column volumes (CV) of DDW filtered water. Equilibrated the column with 5 CV of buffer D. </li>
 <li>Wash the injection loop with DDW (two loop volumes) and repeat with buffer D. Load the protein in the injection loop.</li>
 <li>Inject the protein sample (flow rate of 1 ml/min) and start collecting fractions for SDS-PAGE detection of unbounded proteins.</li>
 <li>If the initial protein volume (step 3.23) is larger than injection loop, repeat steps 3.25-3.26, without washing the loop, until the whole sample is loaded on column. </li>
 <li>Wash the column with 3 CV of buffer C in order to remove unbounded proteins.</li>
 <li>Elute the protein with a 60 min linear gradient of 40 2000mM NaCl between buffers D &amp; E (when 100% buffer E is the end of the gradient), flow rate 1 ml/min (although higher flow rates can be used in the cost of larger volumes, but less concentrated, protein peaks). Collect proteins peaks as they appear in the 280nm chromatogram.</li>
 <li>Clean pre-packed column according to manufacture recommendation. </li>
 <li>SDS-PAGE analysis of collected fractions. Such analysis should be performed with the following samples, unbounded proteins flow-through and protein peaks flow-through. Run the SDS-PAGE and stain as mention in steps 3.17-3.18.</li>
 <li>Evaluate gel for protein bands existence at the predicted Mw according to the protein pre-stained marker. Merge selected elution fractions that contain the relevant protein.</li>
 <li>Dialyze the merged samples, in order to lower NaCl concentrations needed for size exclusion chromatography. Perform the dialysis as mentioned in steps 3.21-3.23 against buffer F.</li>
 <li>Concentrate the protein sample using a Vivaspin-15 10,000 MWCO to 8 mg/ml in a table centrifuge, 4000 rpm. Validate desire concentration by measuring OD at 280nm in Quartz cuvette. If the protein is too concentrated dilute it with buffer F and measure again.</li>
 <li>Assemble size exclusion pre-packed column, HiLoad 26/60 Superdex 200, on FPLC; wash the column with 3 CV of DDW filtered water. Equilibrated the column with 3 CV of buffer F.</li>
 <li>Wash injection loop with 5ml DDW (two loop volumes) followed by the same volume with buffer F. Load no more than 4 ml of the protein sample in the injection loop.</li>
 <li>Inject the protein sample (flow rate of 3.5 ml/min) and start collecting proteins peak fractions as they appear it the 280nm chromatogram. Let the buffer flow until it reaches column volume.</li>
 <li>If the initial protein volume (step 3.23) is larger than injection loop, repeat steps 3.25-3.26, without washing the loop, until the whole sample is loaded on column.</li>
 <li>Clean pre-packed column according to the manufacture recommendation. </li>
 <li>SDS-PAGE analysis of collected fractions. Such analysis should be performed with the protein peaks samples. Run the SDS-PAGE and stain as mention in steps 3.17-3.18.</li>
 <li>Evaluate gel: if protein peaks are specific, in the correct predicted Mw according to the protein pre-stained marker and only one band is visible merge selected eluted fractions of the relevant protein.</li>
 <li>Concentrate protein sample using a Vivaspin-15 10,000 MWCO to &gt;20 mg/ml in a table centrifuge, 4000 rpm. Validate desire concentration by measuring OD at 280nm. The purified MamA&Delta;41 was then concentrated to 26.5 mg/ml for crystallization.</li>
 <li>Confirm sample purity and protein identification using Matrix-assisted laser desorption/ionization (MALDI-TOF).</li>
 <li>If the protein is purified according to the mass spectrometer and SDS-PAGE, divide the concentrated MamA&Delta;41 to pre-marked eppendorf tubes; 25-50&mu;l protein in each.</li>
 <li>Flash frozen in liquid nitrogen and store at 193&deg;K. </li>
</ol>
4. Representative Results
When this protocol is done correctly one should get highly purified, size homogenous and concentrated protein samples. These protein samples are then ready for crystallization trials as well as biochemical studies, such as enzyme kinetics, binding affinity and more. Described here are representative results of main steps in the purification protocol. SDS-PAGE analysis of elution profile of Ni-NTA affinity column should reveal highly over expressed protein in the soluble fraction at appropriate MW of ~22kDa (Figure 1). This SDS-PAGE analysis should also reveal minimum if any protein in the unbounded proteins flow-through, wash flow-through and ultra-centrifugation pellet sample. If large bands of the appropriate protein do appear in these samples one should consider wrong buffer preparation, problems in cell disruption or problems in culture auto-induction conditions and growth.
Ion exchange chromatography is preformed in order to separate between our desirable protein to other E.coli proteins that were bound to the nickel resin (due to electrostatic interactions or histidine/negative amino acids rich protein loops) and uncut His-Tagged desirable protein. This column separates proteins according to their binding affinity to the positively charged resin under increasing NaCl concentrations. Highly negatively charged protein will elute in higher NaCl concentrations appose to moderate negatively charged ones. The advantages of this column are high flow rate and binding capacity. The ion exchange chromatogram (Figure 2) reveals a good separation between 3 proteins populations in increasing NaCl concentration. SDS-PAGE analysis is needed in order to determine MW of each population, isolating the desirable one and evaluation whether further purification steps are needed. The first population is MamA&Delta;41 (~20 kDa) while the second population is MamA&Delta;41+His Tag (~22kDa) that was not cleaved by bovine thrombin and the third population is undetectable in SDS-PAGE due to low concentration. If the proteins peaks are not separated clearly one should consider wrong buffer preparation or changing the slope of NaCl gradient.
Size exclusion chromatography is preformed in order to separate between our desirable protein to other E.coli cell proteins that were bound to the Ni-NTA resin and were not separated during ion exchange chromatography. This column separates proteins according to their size. Large proteins will elute in smaller elution volumes opposed to small ones which will be eluted in larger volumes. The column chromatogram (Figure 3) reveals a good separation of the desirable protein as one main population. The population appears to elute in appropriate monomer size with a MW of ~20 kDa. The presence of ~80 kDa band (E.coli typical protein that binds Ni-NTA resin) is detected in SDS-PAGE prior to column loading and disappears during this run. Since the concentration of this ~80 kDa protein is low to begin with, its population does not appear in the chromatogram due to its dilution and SDS-PAGE analysis is needed to determine the purification efficiency. We recommend loading a diluted and concentrated sample of the main peak to the SDS-PAGE so one could surely determine the presence of a pure protein in the appropriate MW. By this stage the protein is purified and its homogeneity should be evaluated by MALDI-TOF. This analysis revealed a protein in MW of 20347 Da and another in Mw of 10176 Da (Figure 4). The ~10 kDa protein is the ~20 kDa protein doubled charged. The predicted MW of MamA&Delta;41 with the 9 amino acids left after His-Tag removal was 20596.5 Da. By comparing it to the obtained MALDI-TOF MW we found that they are 248 Da apart. This dissimilarity can result from MALDI-TOF measurement errors and/or due to common degradation of the first methionine and the second glycine. To conclude, the protein is highly purified and can be used for further experiments.
<img src="/files/ftp_upload/1844/1844table.jpg" alt="Table 1" /> Table 1. Buffer formulations.
<img src="/files/ftp_upload/1844/1844fig1.jpg" alt="Figure 1" /> Figure 1. Representative SDS-PAGE analysis of Ni-NTA column purification. From right to left; P- ultra-centrifuge pellet (3.11 protocol step), W- wash (3.12 protocol step), U- unbounded proteins (3.10 protocol step), E-five lanes of elution samples (3.14 protocol step), M - protein marker (numbers indicate MW). Arrow indicates MamA&Delta;41.
<img src="/files/ftp_upload/1844/1844fig2.jpg" alt="Figure 2" /> Figure 2. Analysis of ion exchange chromatography step (a) Ion exchange (MonoQ column) chromatogram; Blue - absorption 280nm,
represents protein concentration, Green- NaCl concentration, Red - indication of fractions collected. (b) Representative SDS-PAGE analysis of the ion exchange purification step. From left to right; M - protein marker (numbers indicate Mw), A6 - first peak fraction, A8- second peak fraction.
<img src="/files/ftp_upload/1844/1844fig3.jpg" alt="Figure 3" /> Figure 3. Size exclusion chromatography analysis (a) Size exclusion (Superdex 200 column) chromatogram; Blue - absorption 280nm, represents protein concentration, Red indication of fractions collected. (b) Representative SDS-PAGE analysis of size exclusion purification step. From left to right; M- protein marker (numbers indicate MW), PreI- pre-injected sample, A4-6 combined fraction collected from the first peak, A4-6D- Diluted fraction collected from the peak. 
<img src="/files/ftp_upload/1844/1844fig4.jpg" alt="Figure 4" /> Figure 4. Matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrum of purified MamA&Delta;41. The matrix is Sinapic acid (SA). MamA&Delta;41 shown at 20347 Da, also shown is the doubled charged species of MamA&Delta;41 at 10176 Da.

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Protein purification is the main step in any proteins biochemical or structural studies. Since each protein is unique with its own behavior, one needs to define its properties and modify its purification accordingly. Protein target should be analyzed as a first step toward purification using bioinformatics tools. They are used to calculate the target iso-electric point, assess its need for reducing/oxidizing environment and its need for special ions/ligands. There are several critical modifications of our protocol which ...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French Press</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-078A</td>
<td>&nbsp;</td>
<tr>
<td>Pressure cell</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-032</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge</td>
<td>Equipment</td>
<td>Sorvall</td>
<td>Discovery 90SE</td>
<td>&nbsp;</td>
<tr>
<td>Rottor</td>
<td>Equipment</td>
<td>Beckman</td>
<td>Ti60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td>
<td>Equipment</td>
<td>Beckman</td>
<td>BC-355618</td>
<td>&nbsp;</td>
<tr>
<td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td>
<td>Equipment</td>
<td>BioRad</td>
<td>737-2521</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA His Bind resin</td>
<td>Equipment</td>
<td>Novagen</td>
<td>M0063428</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer</td>
<td>Equipment</td>
<td>Amersham Biosiences</td>
<td>Ultraspec 2100 pro</td>
<td>&nbsp;</td>
<tr>
<td>Quartz cuvette</td>
<td>Equipment</td>
<td>Hellma</td>
<td>104-QS</td>
<td>&nbsp;</td>
<tr>
<td>Fast Performance Liquid Chromatography- AKTA purifier 10</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>28-4062-64</td>
<td>&nbsp;</td>
<tr>
<td>Ion exchange column &#x2013; MonoQ 4.6/100 PE</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>10025543</td>
<td>&nbsp;</td>
<tr>
<td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>17-1071-01</td>
<td>&nbsp;</td>
<tr>
<td>Centricon - Vivaspin15 &#x2013; 10,000 MWCO</td>
<td>Equipment</td>
<td>Sartorius Stedim Biotech GmbH</td>
<td>VS1501</td>
<td>&nbsp;</td>
<tr>
<td>Table centrifuge</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>IEC CL30R</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>Equipment</td>
<td>Bruker Daltonics</td>
<td>Reflex IV</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl (hydrotymethyl) aminomethane</td>
<td>Reagent</td>
<td>BioLab</td>
<td>20092391</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>FRUTROM</td>
<td>235553470</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>Reagent</td>
<td>Alfa Aesar</td>
<td>288-32-4</td>
<td>&nbsp;</td>
<tr>
<td>EDTA free protease inhibitors cocktail</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-8849</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I (Deoxyribonuclease I)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>DN-25</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Thrombin</td>
<td>Reagent</td>
<td>Fisher BioReagents</td>
<td>BP25432</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>BioLab</td>
<td>07132391</td>
<td>&nbsp;</td>
<tr>
<td>Soudim Dodecyl Sulfate (SDS)</td>
<td>Reagent</td>
<td>BioLab</td>
<td>19822391</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-3148</td>
<td>&nbsp;</td>
<tr>
<td>InstantBlue</td>
<td>Reagent</td>
<td>Expedeon</td>
<td>1SB01L</td>
<td>&nbsp;</td>
<tr>
<td>PageRuler Prestained Protein Ladder</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>SM0671</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÎ”41 at JoVE.com

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.

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 ...

purification-m-magneticum-strain-amb-1-magnetosome-associated-protein

Research

JoVE Journal

Biology

13.5K 次观看.  Ben-Gurion University. 妈妈是一个独特的磁相关蛋白被证明是参与磁激活。在这里，我们从目前的妈妈缺失突变体的净化协议（MamAΔ41） M。 magneticum AMB - 1。

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

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 Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

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 ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;