Name: Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA and Delta;41 (Video) | JoVE Journal
Uploaded: 2010-03-25T16:45:00.000+00:00
Duration: 11 min 7 s
Description: Learn the purification protocol for MamAΔ41 from M. magneticum AMB-1 using affinity, ion exchange, and size exclusion chromatography. This method enables structural and functional studies of magnetosome-associated proteins.

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5 minutes

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 mamAΔ41 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 pET52bMamAΔ41-AMB1. In this construct, the mamAΔ41 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 pET52bMamAΔ41 was grown in auto-induction (15) medium containing ampicillin (50 mg/ml) 310°K for 3 hours. The cultivation temperature was then shifted from 310° to 300°K and maintained for an additional 48 h at 300°K. The cells were harvested by centrifugation at 5465g for 10 min at 277°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-MamAΔ41 the MW is 22529 Da (240 amino acids) and for MamAΔ41 (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-MamAΔ41 is 0.595 and for MamAΔ41 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 MamAΔ41
<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 0.22μm 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 10μl 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°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 50μl of 5% β-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 10μl from each sample with 10μl of 5% β-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/μl), 10μl 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°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 MamAΔ41 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 MamAΔ41 to pre-marked eppendorf tubes; 25-50μl protein in each.</li>
 <li>Flash frozen in liquid nitrogen and store at 193°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 MamAΔ41 (~20 kDa) while the second population is MamAΔ41+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 MamAΔ41 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="Buffer preparation table, reagents concentrations: Tris-HCl, NaCl, Imidazole; volumes measured." data-altupdatedat="1788235205000" data-alt="Table 1"> Table 1. Buffer formulations.
<img src="/files/ftp_upload/1844/1844fig1.jpg" alt="SDS-PAGE gel, protein separation, electrophoresis results, molecular weight markers." data-altupdatedat="1788235205000" data-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 MamAΔ41.
<img src="/files/ftp_upload/1844/1844fig2.jpg" alt="Chromatography and SDS-PAGE gel; protein analysis; size separation and molecular weight determination." data-altupdatedat="1788235205000" data-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="Protein purification; chromatography graph and SDS-PAGE gel for protein analysis result comparison." data-altupdatedat="1788235205000" data-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="Mass spectrometry graph showing ion intensity peaks; spectrum analysis; mass-to-charge ratio data." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrum of purified MamAΔ41. The matrix is Sinapic acid (SA). MamAΔ41 shown at 20347 Da, also shown is the doubled charged species of MamAΔ41 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 ...

<table><tbody><tr><td>French Press</td><td>Equipment</td><td>Thermo Fisher Scientific, Inc.</td><td>FA-078A</td><td></td></tr><tr><td>Pressure cell</td><td>Equipment</td><td>Thermo Fisher Scientific, Inc.</td><td>FA-032</td><td></td></tr><tr><td>Ultra-centrifuge</td><td>Equipment</td><td>Sorvall, Thermo Scientific</td><td>Discovery 90SE</td><td></td></tr><tr><td>Rottor</td><td>Equipment</td><td>Beckman Coulter Inc.</td><td>Ti60</td><td></td></tr><tr><td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td><td>Equipment</td><td>Beckman Coulter Inc.</td><td>BC-355618</td><td></td></tr><tr><td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td><td>Equipment</td><td>Bio-Rad</td><td>737-2521</td><td></td></tr><tr><td>Ni-NTA His Bind resin</td><td>Equipment</td><td>Novagen, EMD Millipore</td><td>M0063428</td><td></td></tr><tr><td>Spectrophotometer</td><td>Equipment</td><td>Amersham</td><td>Ultraspec 2100 pro</td><td></td></tr><tr><td>Quartz cuvette</td><td>Equipment</td><td>Hellma</td><td>104-QS</td><td></td></tr><tr><td>Fast Performance Liquid Chromatography- AKTA purifier 10</td><td>Equipment</td><td>GE Healthcare</td><td>28-4062-64</td><td></td></tr><tr><td>Ion exchange column &amp;#8211; MonoQ 4.6/100 PE</td><td>Equipment</td><td>GE Healthcare</td><td>10025543</td><td></td></tr><tr><td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td><td>Equipment</td><td>GE Healthcare</td><td>17-1071-01</td><td></td></tr><tr><td>Centricon - Vivaspin15 &amp;#8211; 10,000 MWCO</td><td>Equipment</td><td>Sartorius AG</td><td>VS1501</td><td></td></tr><tr><td>Table centrifuge</td><td>Equipment</td><td>Thermo Fisher Scientific, Inc.</td><td>IEC CL30R</td><td></td></tr><tr><td>MALDI-TOF</td><td>Equipment</td><td>Bruker Corporation</td><td>Reflex IV</td><td></td></tr><tr><td>Tris-HCl (hydrotymethyl) aminomethane</td><td>Reagent</td><td>BioLab</td><td>20092391</td><td></td></tr><tr><td>Sodium Chloride</td><td>Reagent</td><td>FRUTROM</td><td>235553470</td><td></td></tr><tr><td>Imidazole</td><td>Reagent</td><td>Alfa Aesar</td><td>288-32-4</td><td></td></tr><tr><td>EDTA free protease inhibitors cocktail</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P-8849</td><td></td></tr><tr><td>Dnase I (Deoxyribonuclease I)</td><td>Reagent</td><td>Sigma-Aldrich</td><td>DN-25</td><td></td></tr><tr><td>Bovine Thrombin</td><td>Reagent</td><td>Fisher Scientific</td><td>BP25432</td><td></td></tr><tr><td>Glycine</td><td>Reagent</td><td>BioLab</td><td>07132391</td><td></td></tr><tr><td>Soudim Dodecyl Sulfate (SDS)</td><td>Reagent</td><td>BioLab</td><td>19822391</td><td></td></tr><tr><td>Beta-mercapt&amp;#8211;thanol</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M-3148</td><td></td></tr><tr><td>InstantBlue</td><td>Reagent</td><td>Expedeon</td><td>1SB01L</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder</td><td>Reagent</td><td>Fermentas</td><td>SM0671</td><td></td></tr></tbody></table>

purification of the m. magneticum strain amb-1 magnetosome associated protein mama&delta;41

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

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

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

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

The purification process begins with cell pellets expressing the Mame. Delta 41 recombinant protein cell membrane disruption is followed by separation of the soluble and insoluble fraction. In order to extract the soluble recombinant protein, affinity chromatography separates the tagged recombinant protein from other cellular proteins.Ion exchange chromatography separates mamma Delta 41 from other cellular proteins that remain after affinity chromatography size exclusion. Chromatography further separates mamma Delta 41 and reveals its oligomeric state. Finally, the recombinant protein purity and homogeneity is analyzed.Hi, I'm Raza, the head of the XVA Laboratory in the Life Sciences Department at the Benon University of the Negative. I'm Natalie also from Z Lab. Today we will show you a procedure for recombinant protein purification.We use this procedure in our laboratory to study magnetism associated proteins. So let's get started At the start of this protocol. The MAME Delta 41 protein is expressed as a 10 hiss fusion protein.In BL 21 e coli cells grow the cells in auto induction media containing Epi Brazilin at 310 K for three hours. Then shift the cultivation temperature to 300 K and allow the cells to incubate for 48 hours. Following incubation, harvest the cells by centrifugation, decant the supernatant and weigh the resulting cell pellet.Eight liters of culture should produce about 60 grams of wet cell pellet. Prior to protein purification. Prepare 200 milliliter stalk solutions of four molar ile, five molar sodium chloride, and one molar tris H-C-L-P-H eight.Using double distilled water or DDW vortex the solutions until clear and filter them with a point 22 micrometer. Filter from the stock solutions freshly prepared and filter buffers A through F as described in the written protocol accompanying this video. To begin protein purification, re suspend the cells in buffer A in a one to two ratio of cell mass to buffer volume.For every 10 grams of bacteria, add 10 microliters of dna, one to digest any DNA fragments in the sample. Then add one milliliter of E dt, a free protease inhibitor cocktail. For every 20 grams of bacteria, incubate the solution for 20 minutes on ice.Following incubation, lyce the cells with two cycles of French press at 25, 000 PSI. Due to temperature buildup, the piston should be cooled on ice for 10 minutes prior to use. Transfer the lysed cells into 25 milliliter ultracentrifuge tubes.Fill the tubes to the neck line with buffer A and balance them precisely. Then separate the cell debris by ultracentrifugation. Meanwhile, assemble a nickel NTA gravity column by adding four milliliters of nickel NTA resin to a 2.5 centimeter glass column.Prepare the resin for protein purification according to the manufacturers and S instructions. Once the samples have been centrifuged, pour the soluble fraction from the ultra centrifugation tubes over the nickel NTA column. Collect the unbound proteins in the flow through fraction.Continue to wash the column with 100 milliliters of buffer B and collect the flow through. Preserve the flow through fractions at 277 K.Elude the protein in 15 numbered 1.5 milliliter einor tubes. Close the column valve and load one milliliter of buffer C onto the column.After a three minute incubation time, open the valve and collect the flow through. Repeat this step three times. Continue to elude the protein at one milliliter buffer increments without the three minute incubation.Using a UV spectrophotometer, locate the protein peak in the fractions by measuring the optical density at 280 nanometers. Finally, perform an SDS page analysis of the collected fractions. The unbound protein flow through the wash, flow through and an ultracentrifugation pellet sample from the gel.Determine which eluded fractions have high protein levels merge the selected fractions. To remove the 10 hiss tag, add 10 microliters of bovine thrombin per one milligram of protein to the combined sample. Next, dialyze the sample against buffer D to remove the excess in midol and decrease the sodium chloride concentration.In preparation for ion exchange chromatography, cut the desired length of seven kilodalton molecular weight cutoff dialysis tubing. Wash the tubing with DDW and make a bag by tying a knot At one end, transfer the thrombin digested protein solution into the bag, leaving some space at the top. Clamp the dialysis tubing closed.Submerge the dialysis tubing into four liters of chilled buffer D.Stir the solution slowly overnight at 277 K after dialyzing overnight, remove the tubing from the beaker. Make a small cut and transfer the protein to a 50 milliliter Falcon tube ion exchange chromatography is used to further purify the memay Delta 41 protein. Connect a pre-packed mono Q4 0.6 100 PE column to an FPLC ACTA purifier system.Wash the column with five column volumes or CVS of filter DDW equilibrate the column with five cvs of buffer D.Wash the injection loop with two loop volumes of DDW followed by two loop volumes of buffer D.Load the protein into the injection loop. Inject the protein sample at a flow rate of one milliliter per minute and begin collecting fractions. That will be used for SDS page detection of unbound proteins.Continue to load and inject the protein until the entire sample is loaded onto the column. Wash the column with three cvs of buffer D.To remove any unbound proteins, elude the protein over 60 minutes at one milliliter per minute using a linear gradient of sodium chloride starting at 40 millimolar and ending at two molar. Collect the protein peaks as they appear in the 280 nanometer chromatogram after 100%buffer E has been reached.Clean the prepec column according to manufacturers recommendation. Perform SDS page analysis of the collected FPLC fractions including the unbound protein and protein peak flow through using prestained markers. Evaluate the protein bands to identify the fractions containing protein at the predicted molecular weight of Mame.Delta 41 merge the selected fractions in preparation for size exclusion chromatography. The sodium chloride concentration of the merged samples is lowered by dialyzing as previously described against buffer f. After overnight dialysis.Use a tabletop centrifuge to concentrate the protein sample using a centrifugal concentrator with a 10, 000 molecular weight cutoff. Concentrate the protein to eight milligrams per milliliter at 4, 000 RPM. Validate the desired concentration by measuring the OD at 280 nanometers in a quartz vete.Assemble the high load 26 60 superdeck 200 size exclusion pre-packed column on an FPLC ACTO purifier system. Wash the column with three cvs of filtered water and equilibrate the column with three cvs of buffer F.Wash the injection loop with two loop volumes of DDW followed by the same volume of buffer F.Once the FPLC has been washed and equilibrated, load the protein sample into the injection loop. Inject the protein sample and start collecting protein peak fractions as they appear in the 280 nanometer chromatogram.Let the buffer flow until the column void volume is reached. Clean the pre-packed column according to the manufacturer's recommendation. Perform SDS page analysis of the collected fractions and evaluate the gel.The protein should appear at the correct predicted molecular weight according to the protein pre-sent marker and only one band should be visible. Merge the pure protein fractions and concentrate the protein sample to greater than 20 milligrams per milliliter using a centrifugal concentrator with a 10, 000 molecular weight cutoff and a tabletop centrifuge. Validate the desired concentration by measuring the OD at 280 nanometers.Confirm the MAME delta 41 sample purity and identity using matrix assisted laser desorption ionization formal D tof. Finally divide the concentrated purified MAME delta 41 protein into pre-marked eend orph tubes at a volume of 25 to 50 microliters each. The sample can be flash frozen in liquid nitrogen and stored at 193 K.This protocol should result in a highly pure size homogenous concentrated MAME Delta 41 protein sample SDS page analysis of the nickel NTA elution profile reveals high levels of memay delta 41 10 hiss protein in the elution fractions at approximately 22 kilodaltons.The SDS page analysis will also reveal any memay delta 41 protein in the unbound protein flow through the wash, flow through and the ultracentrifugation pellet sample. If large bands around 22 kilodaltons do appear in these samples, troubleshooting is necessary. Ion exchange chromatography is performed to separate the MAME Delta 41 protein from other proteins in the sample through ion exchange.Under increasing sodium chloride concentrations, the ion exchange chromatogram reveals good separation between two populations of proteins under increasing sodium chloride. Concentration analysis of the samples by SDS page reveals the molecular weight of each population. The first peak is MAME delta 41, while the second peak is the MAME delta 41 10 hiss protein that was not cleaved by bovine thrombin.If ion exchange chromatography does not result in fully resolved protein peaks, troubleshoot buffer preparation and the slope of the sodium chloride gradient. The final step of the MAME delta 41 purification is size exclusion chromatography. The chromatogram reveals a single peak eluding at 20 kilodaltons.The appropriate monomer size of the MAME Delta 41 protein maldi to is done to verify the purified MAME delta 41 protein. This analysis reveals two species, one representing the ly charged protein at 20, 347 Daltons and the other representing the doubly charged protein at 10, 176 Daltons. The predicted molecular weight for the recombinant mam MA delta 41 protein following hist tag removal is 20, 597 Daltons.The small 250 Dalton difference may be due to the common degradation of the first methionine in the second glycine. The results reveal highly purified MAME delta 41 protein. It can be used for further experiments.We've just shown you how to purify a recombinant syllable protein When performing this procedure. It's important to remember to adjust the buffers to your unique proteins and to perform these experiments as fast as possible. So that's it.Thanks for watching and good luck with your experiments.

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