purification of self-assembling protein nanoparticles using affinity chromatography

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

For the lysis of harvested E. coli expressing the six-helix bundle SAPN, use a probe with a sonication output of 150 watts, with four seconds of sonication and six seconds of rest to sonicate resuspended bacterial pellets in 40 milliliters of imidazole-free buffer on ice for five minutes.
Centrifuge the cellular lysate to generate a clarified supernatant, and transfer the supernatant to a sterile 150-milliliter flask. Then, bring the sample up to a final volume of 100 milliliters with fresh imidazole-free buffer.
To isolate the protein of interest, open the FPLC software and click the New Method option. In the Method Settings menu, open the Column Position dropdown menu and select C1 Port 3. In the Shown By Technique dropdown menu, select Affinity. In the Column Type dropdown menu, select Others, HisTrap HP, and five milliliters. The column volume and the pressure boxes will automatically be set to the appropriate values.
Click Method Outline and drag the Equilibration and Sample Application buttons, three Column Wash buttons, and the Elution button from the Phase Library pop-up menu next to the arrow, before closing the Phase Library menu. Click Equilibration and set the values listed in the table to Initial Buffer B, 4%, Final Buffer B, 4%, and Column Volume, 5.
Click Sample Application. In the Sample Loading box, click the Radio button for Inject Sample on Column with Sample Pump and confirm that the box next to the Use Flow Rate From Method Settings is checked in the Sample Injection with System Pump Box. Change the Volume box value to 100 milliliters and the Fraction Collection Scheme to Enable.
Unclick the Use Fraction Size from Method Settings box and change the fraction size to four milliliters. Click on the first Column Wash button. Set the values listed in the table to Initial Buffer B, 4%, Final Buffer B, 4%, and Column Volume, 10. Click the Fraction Collection Scheme Enable box and unclick the Use Fraction Size for Method Settings. Change the fraction size to four milliliters and click the second Column Wash button.
Set the values in the table to Initial Buffer B, 0%, Final Buffer B, 0%, and Column Volume, 5. Click the Fraction Collection Scheme Enable box and unclick the Use Fraction Size from Method Settings. Change the fraction size to 4 milliliters and click the Third Column Wash button. Set the values in the table to Initial Buffer B, 0%, Final Buffer B, 0%, and Column Volume, 10. Click the Fraction Collection Scheme Enable button and unclick the Use Fraction Size for Method Settings box. Then, change the fraction size to 4 milliliters.
Click the Elution button and right-click the information in the table. In the pop-up menu, click Delete Step, and drag the Isocratic Gradient button onto the table two times so that there are two entries. Set the value for the first entry to Initial Buffer B, 30%, Final Buffer B, 30%, and Column Volume, 10.
Set the value for the second entry to Initial Buffer B, 100%, Final Buffer B, 100%, and column volume, 10. Click the Fraction Collection Scheme Enable button, and click the Use Fraction Size for Method Settings box. Then, click Save As and name the file, Purification.
Place the pump A tubing of the FPLC into the imidazole-free wash buffer, and the pump B tubing into the 500-millimolar imidazole buffer. Place the sample pump tubing into the 100-milliliter sample and run the purification program. When the 60% isopropanol wash is needed, pause the program, move the pump tubing from the imidazole-free wash into the 60% isopropanol wash, and restart the program. At the end of the wash, pause the program again, return the pump A tubing to the imidazole-free wash buffer, and restart the purification program.

purification-self-assembling-protein-nanoparticles-using-affinity

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1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)
<ol>
	<li>Mix 95 mL of component A and 5 mL of component B of the media in a 2 L sterile glass Erlenmeyer flask as per the manufacturer&#39;s instructions (see the Table of Materials). Add ampicillin to a final concentration of 100 &#956;g/mL.</li>
	<li>Inoculate the media with E. coli from a previously established glycerol stock culture. Incubate culture at 30 &#176;C with shaking at 200 rotations per minute (rpm) for 48 h. 
	NOTE: The used E. coli BL21 (DE3) stock contained the ampicillin resistant expression vector with the SHB-SAPN gene. Although the general protocol of the media recommends 24 h of incubation at 37 &#176;C, 48 h of incubation at 30 &#176;C gives higher yield for SHB-SAPN.</li>
	<li>Transfer the culture to two 50 mL conical tubes. Centrifuge the tubes at 4,000 x g for 10 min with a fixed angle rotor at 4 &#176;C. Remove the supernatant and save the pellet to harvest cells. 
	NOTE: The cell pellet can be either processed immediately or frozen at -80 &#176;C until use.</li>
</ol>
2. Lysis of E. coli BL21(DE3) by sonication
NOTE: Use nonpyrogenic plasticware and glassware baked at 250 &#176;C for at least 30 min. Tris(2-carboxyethyl)phosphine (TCEP), as a reducing agent, breaks the disulfide bonds within and between proteins. TCEP is necessary for the buffers during this protocol if the displayed antigen contains S-S bonds. For the SHB-SAPN core only, the presence of TCEP in the buffers is not essential.
<ol>
	<li>Prepare imidazole-free buffer (8 M Urea, 50 mM sodium phosphate monobasic, 20 mM Tris base, 5 mM TCEP) pH 8.0 (adjusted with 5 N sodium hydroxide, NaOH) and filter it using a 0.22 &#181;m vacuum bottle filtration unit.</li>
	<li>Resuspend the pelleted cells (from step 1.3) with 40 mL of imidazole-free buffer in one 50 mL conical tube. Sonicate the resuspended cells with a probe on ice for 5 min (4 s of sonication, 6 s of rest) with a sonication output of 150 W.</li>
	<li>Centrifuge the cellular lysate (40 mL) at 29,000 x g at 4 &#176;C for 25 min in a fixed angle rotor to generate clarified supernatant. Transfer the supernatant to a 150 mL sterile flask and discard the pellet. Dilute the supernatant to 100 mL using the imidazole-free buffer (later in the protocol referred to as &#34;sample&#34;). 
	NOTE: This dilution step is needed to prevent the fast performance liquid chromatography (FPLC) system pressure from becoming too high during lysate loading on the column.</li>
</ol>
3. Protein purification using a His-column
NOTE: This protocol was performed using an FPLC instrument, but it can be adapted to gravity flow.
<ol>
	<li>Prepare the following buffers and filter them using a 0.22 &#181;m vacuum bottle filtration unit: (i) imidazole-free buffer &#34;Buffer A&#34; (8 M Urea, 50 mM sodium phosphate monobasic, 20 mM Tris base, 5 mM TCEP) pH 8.0; (ii) 500 mM imidazole buffer &#34;Buffer B&#34; (8 M Urea, 50 mM sodium phosphate monobasic, 20 mM Tris base, 5 mM TCEP, 500 mM Imidazole) pH 8.0; and (iii) isopropanol wash (20 mM Tris, 60% isopropanol) pH 8.0. 
	NOTE: pH for each buffer was adjusted with 5 N NaOH.</li>
	<li>Equilibrate the His-column. 
	NOTE: For lab scale production, this protocol uses a 5 mL prepacked His-column, but any larger sized column can be used.
	<ol>
		<li>Open the FPLC software and click on the New method option. It will immediately open to the Method settings menu. Under the drop-down menu for column position choose C1 port 3.</li>
		<li>On the Shown by technique drop down menu choose affinity. On the Column type drop-down menu choose others, Histrap HP, 5 mL. The column volume and the pressure boxes will be automatically set to the appropriate values.</li>
		<li>Click on the Method outline button. Drag the following buttons from the Phase library popup menu: equilibration, sample application, column wash, and elution next to the arrow in that exact order. Close the Phase library menu.</li>
		<li>Click on the equilibration button. The values listed in the table should be &#34;initial buffer B&#34; (4%), &#34;final buffer B&#34; (4%), and &#34;volume (CV)&#34; 5.</li>
		<li>Click on the Sample application box. In the sample loading box click the radio button for Inject sample on column with sample pump. Make sure the box next to the Use flow rate from method settings is checked in the sample injection with system pump box. Next to the volume box on the right side of the screen change the value to 20 mL.</li>
		<li>Click on the Column wash button. The values listed in the table should be &#34;initial buffer B&#34; (4%), &#34;final buffer B&#34; (4%), and &#34;volume (CV)&#34; 5. Next to the fraction collection scheme unclick the Enable box.</li>
		<li>Click on the elution button. The values listed in the table should be &#34;initial buffer B&#34; 0%, &#34;final buffer B&#34; 100%, and &#34;volume (CV)&#34; 5. Next to the fraction collection scheme, click on the Enable box. Unclick the Use fraction size from method settings and adjust the fraction size to 4 mL in the fill-in box below.</li>
		<li>Click the save as button on the top of the software. Name the file &#34;equilibration.&#34;</li>
		<li>Connect a 5 mL prepacked His-column to the corresponding column port 3 on the FPLC. Both pump A and pump B tubing, as well as the sample pump tubing, should be placed into 0.22 &#181;m filtered deionized water. Run the equilibration program.</li>
		<li>Place both pump A and pump B, as well as the sample pump tubing, into the imidazole-free buffer (Buffer A) and run the equilibration protocol again.</li>
	</ol>
	</li>
	<li>Bind the sample to the column and purify the protein.
	<ol>
		<li>Open the FPLC software and click on the New method option. It will immediately open to the Method settings menu. Under the drop-down menu for Column, position choose C1 port 3. On the Shown by technique drop-down menu, choose affinity. On the column type drop-down menu choose others, Histrap HP, 5 mL. The column volume and the pressure boxes will automatically be set to the appropriate values.</li>
		<li>Click on the Method outline button. Drag the buttons from the Phase library popup menu: equilibration, sample application, column wash (Wash 1), column wash (Wash 2), column wash (Wash 3), and Elution next to the arrow in that exact order. Close the Phase library menu.</li>
		<li>Click on the Equilibration button. The values listed in the table should be &#34;initial buffer B&#34; 4%, &#34;final buffer B&#34; 4%, and &#34;volume (CV)&#34; 5.</li>
		<li>Click on the Sample application box. In the sample loading box, click the radio button for the Inject sample on the column with the sample pump. Make sure the box next to the Use flow rate from method settings is checked in the sample injection with the system pump box.</li>
		<li>Next to the volume box on the right side of the screen, change the value to 100 mL. Next to the fraction collection scheme, click the Enable button. Unclick the Use fraction size from the method settings box and then change the fraction size to 4 mL.</li>
		<li>Click on the first column wash button (Wash 1). The values listed in the table should be &#34;initial buffer B&#34; 4%, &#34;final buffer B&#34; 4%, and &#34;volume (CV)&#34; 10. Next to the fraction collection scheme, click the Enable box. Unclick the Use fraction size from method settings and then change the fraction size to 4 mL.</li>
		<li>Click on the second column wash button (Wash 2). The values listed in the table should be &#34;initial buffer B&#34; 0%, &#34;final buffer B&#34; 0%, and &#34;volume (CV)&#34; 5. Next to the fraction collection scheme, click the Enable box. Unclick the Use fraction size from method settings and then change the fraction size to 4 mL.</li>
		<li>Click on the third column wash button (Wash 3). The values listed in the table should be &#34;initial buffer B&#34; 0%, &#34;final buffer B&#34; 0%, and &#34;volume (CV)&#34; 5. Next to the fraction collection scheme, click Enable. Unclick the Use fraction size from the method settings box and then change the fraction size to 4 mL.</li>
		<li>Click on the Elution button. In the table right, click the information listed and, on the menu that comes up, click the Delete step. Drag the isocratic gradient button onto the table twice so that there are two entries.</li>
		<li>The value for the first entry should read &#34;initial buffer B&#34; 30%, &#34;final buffer B&#34; 30%, and &#34;Volume (CV)&#34; 10. The value for the second entry should read &#34;initial buffer B&#34; 100%, &#34;final buffer B&#34; 100%, and &#34;volume (CV)&#34; 10. Next to the fraction collection scheme, click the Enable button. Click the box next to Use fraction size from method settings.</li>
		<li>Click the Save button on the top of the software. Name the file &#34;purification.&#34; Pump A tubing of the FPLC should be placed into the imidazole-free wash buffer, while pump B tubing should be placed into the 500 mM imidazole buffer. The sample pump tubing should be placed into the 100 mL sample.</li>
		<li>Run the &#34;purification&#34; program and wait for the time when the 60% isopropanol is needed (Wash 2). Pause the program; move pump A tubing from the imidazole-free wash into the 60% isopropanol wash. Restart the program.</li>
		<li>Once the isopropanol step is completed, pause the program again and move to pump A tubing back into the imidazole-free wash buffer. Restart the purification program (the rest of the run is automated).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-propanol</td>
			<td>Fisher</td>
			<td>BP26181</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher</td>
			<td>BP1760-25</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Corning Disposable Vacuum Filter/Storage Systems</td>
			<td>Fisher</td>
			<td>09-761-108</td>
			<td>A variety of sizes</td>
		</tr>
		<tr>
			<td>GE Healthcare 5 mL HisTrap HP Prepacked Columns</td>
			<td>GE HealthCare</td>
			<td>45-000-325</td>
			<td>5 pack</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-4</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Fisher</td>
			<td>O3196-500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Magic Media E. coli Expression Medium</td>
			<td>ThermoFisher</td>
			<td>K6803</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>MilliporeSigma Millex Sterile Syringe 0.22 mm Filters</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td>250 pack</td>
		</tr>
		<tr>
			<td>One Shot BL21 Star (DE3) Chemically Competent E. coli</td>
			<td>ThermoFisher</td>
			<td>C601003</td>
			<td>20 vials</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Fisher</td>
			<td>BP329-500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher</td>
			<td>BP152-1</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Fisher</td>
			<td>BP169-500</td>
			<td>2.5 kg</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiberlite F14-14 x 50cy Fixed-Angle Rotor</td>
			<td>ThermoFisher</td>
			<td>096-145075</td>
			<td>Rotor</td>
		</tr>
		<tr>
			<td>NGC Quest 10 Chromatography System</td>
			<td>BioRad</td>
			<td>7880001</td>
			<td>FPLC to aid in protein purification</td>
		</tr>
		<tr>
			<td>Sonifer 450</td>
			<td>Branson</td>
			<td>also known as 096-145075</td>
			<td>Sonicator</td>
		</tr>
	</tbody>
</table>

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

Take a suspension of engineered bacteria expressing recombinant self-assembling protein nanoparticles with polyhistidine tags.
Sonicate to release intracellular components.
Centrifuge to collect the supernatant containing protein nanoparticles. Dilute it with an imidazole-free buffer.
Take an affinity chromatography column containing agarose beads with immobilized nickel cations.
Add a buffer with a low imidazole concentration for column equilibration.
Add the bacterial lysate onto the column.
During the run, the polyhistidine on protein nanoparticles binds strongly with nickel cations.
Meanwhile, contaminants like bacterial lipopolysaccharides and other proteins bind weakly to the column.
Wash with a buffer containing a low imidazole concentration to remove the weakly bound proteins.
Introduce isopropanol to remove lipopolysaccharides and then wash.
Next, introduce a buffer with an intermediate imidazole concentration. The imidazole competes with histidine for binding to the nickel cation, releasing bound protein nanoparticles.
Next, add a buffer containing a high imidazole concentration to completely elute the protein nanoparticles.
Collect the purified protein nanoparticles for further application.

43 Views. Purificazione di nanoparticelle proteiche autoassemblanti mediante cromatografia di affinità

Video: Purificazione di nanoparticelle proteiche autoassemblanti mediante cromatografia di affinità - Experiment

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

Sintesi e caratterizzazione di 1,2-Dithiolane per volta peptidi autoassemblanti

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

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Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Produzione su scala libera da cellule e aggiunta adiuvante a una proteina principale ricombinante della membrana esterna da Chlamydia muridarum per lo sviluppo di vaccini

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Immunologia e infezioni

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificazione e ripiegando a amiloide fibrille di (sua)<sub&gt; 6</sub&gt; -tagged proteina ricombinante Shadoo Espresso come corpi di inclusione in<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

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Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography