Name: Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation (Video) | JoVE Journal
Uploaded: 2019-08-21T15:00:00.000+00:00
Duration: 10 s
Description: Learn to purify, refold, and characterize self-assembling protein nanoparticles for vaccine development using E. coli expression. This method enables trimeric antigen presentation and supports scalable clinical production.

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This work was supported by a cooperative agreement (W81XWH-11-2-0174) between the Henry M Jackson Foundation for the Advancement of Military Medicine, Inc., and the US Department of Defense. The anti-HIV-1 gp41 mAb 167-D IV antibody was received from Dr. Susan Zolla-Pazner through the NIH AIDS Reagent Program.

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The views expressed are those of the authors and should not be construed to represent the positions of the US Army or the Department of Defense. Peter Burkhard has an interest in the company called Alpha-O Peptides AG and has patents on the technology. The other authors have no affiliation or financial involvement with any company with a financial interest on the subject matter presented in this paper.

Nanotechnology provides many advantages and solutions for subunit vaccine development. Nanovaccines can repeatedly present antigens as particulates to the host immune system increasing immunogenicity26. While there are many different types of nanovaccines, we believe that ones composed of de novo designed protein seem to be the strongest approach for vaccine development1. They can be engineered without any sequence homology to the host proteins and present the antigen of interest in close to native-like conformation while providing low production cost and high product yields. A prime example of this approach is the SAPN technology, which we have applied to vaccines against multiple infectious diseases7. Addressing the difficulties in HIV-1 vaccine development, we have engineered a unique SHB-SAPN core to effectively present the V1V2 antigen in a native-like trimeric conformation9. Many vaccine targets, particularly for viral diseases, are present as trimers27. This phenomenon indicates that our SAPN design has wide implications for the development of subunit vaccines.
In this method, we demonstrate how to produce SHB-SAPNs in an E. coli expression system. We expressed high yields of protein (about 6 mg/100 mL of culture). The protein contained 10 histidines and was easily purified using an immobilized metal affinity chromatography with Ni2+ column. This length of the His-Tag was found to be the optimal for the highest protein yield. The purified protein contained the full-length of the designed protein as indicated by the presence of both the N-terminal HisTag and the C terminal heptad repeat. We utilized widely accepted techniques and optimized them for the expression, production, and characterization of the SHB-SAPN core. Lack of the production of the full-length protein during the development of a SAPN containing a new protein epitope could indicate an expression problem of the gene in the host cell. If it happens, the gene and the expression system must be redesigned and adapted to the described protocol. Modification of sonication time or intensity may also increase the concentration of the predicted full-length protein.
Refolded particles were in the expected size range (20 to 100 nm)8,24,25 as determined by DLS and nanoparticle tracking analysis. These results were further confirmed by using TEM. If there are problems in this step, it is normally due to a problem with the pH or ionic strength of the refolding buffer. When large size particles are detected on the particle sizing techniques, it indicates aggregation, which can be avoided by increasing the pH of the refolding buffer. If the particles are not detected by DLS, verify the concentration of the protein and check the pH of the buffer. The final protein concentration for DLS should be at least 100 &#181;g/mL. If the concentration is not the problem, it indicates the abundance of small, incompletely formed particles, whose concentration can be reduced by decreasing the pH. Alternatively, the sodium chloride concentration can be adjusted to the optimum range to minimize the presence of particles with unwanted size.
Finally, by using an isopropanol wash step during purification we were able to reduce contaminating LPS from the host E. coli to 0.01 EU/&#181;g of SAPN which is below the Food and Drug Administration (FDA) limit of 5 EU/kg of body weight for injectable products28. This level can be further reduced by using an anion exchange column also known as Q column. If high levels of endotoxin are still present, check all materials that were used for buffer preparation. Remember to use only depyrogenated glassware and endotoxin free plasticware in this method.
These results indicate that we have successfully developed a method to produce the SHB-SAPN core that can be used for pre-clinical immunization studies. This method with only slight modifications, if any, can be applied to the purification of SHB-SAPNs when an antigen of interest is added. Using this method as a starting point one of the major changes is in the elution step. Different proteins elute at different imidazole concentrations that must be determined experimentally. The other major difference might be the composition of the refolding buffer. Optimization would require testing different pH conditions as well as ionic strengths.
In consideration of future work, only two slight modifications are needed to allow human application of the SHB-SAPN. The first is that the expression vector needs to be changed to a kanamycin resistance selectable marker due to the ampicillin allergy in humans29. The other major requirement of the protein manufacturing for human use is to produce the SHB-SAPN in animal product-free media. A small-scale study already indicated a reasonable yield of protein in a plant-based media. The work presented here is easily scalable for ultimate GMP production as demonstrated with a malaria vaccine candidate, FMP01416. This large scale FMP014-SAPN production included both the anion exchange and the cation exchange steps to further reduce LPS and Ni2+ content from the final product. This bacterial-expressed SAPN has been already scaled up for an upcoming Phase 1/2a clinical trial.

While traditional vaccine development has focused on the inactivated or attenuated pathogens, the focus of modern vaccines has shifted toward subunit vaccines1. This approach can lead to a more targeted response, and potentially more efficacious vaccine candidates. However, one of the main drawbacks is that subunit vaccines are not particulates like whole organisms which can result in reduced immunogenicity2. A nanoparticle as a repetitive antigen display system can have the benefits of both the targeted subunit vaccine approach as well as the particulate nature of the whole organism1,3.
Among the existing types of nanovaccines, rationally designed protein assemblies allow for the design and development of vaccine candidates that can present multiple copies of the antigen potentially in a native-like conformation1,4,5,6. One example of these protein assemblies are the self-assembling protein nanoparticles (SAPNs)7. SAPNs are based on coiled-coil domains and are traditionally expressed in Escherichia coli8. SAPN vaccine candidates have been developed for a variety of diseases such as malaria, SARS, influenza, toxoplasmosis, and HIV-19,10,11,12,13,14,15,16,17,18,19. The design of each SAPN candidate is specific to the pathogen of interest, however, the production, purification, and refolding techniques are generally broadly applicable.
One of our current interests is an effective HIV-1 vaccine. In RV144&#8212;the only Phase III clinical trial of an HIV-1 vaccine that demonstrated modest efficacy&#8212;the reduced risk of infection was correlated with IgG antibodies to the V1V2 loop of the envelope protein20,21. The native-like trimeric presentation of this region is thought to be important for protective immunogenicity22. To present the V1V2 loop in as close to native-like conformation as possible, we developed a proof of principle SAPN vaccine candidate that contained the HIV-1 envelope post-fusion six-helix bundle (SHB) to present the V1V2 loop into the correct conformation9. This candidate was recognized by known monoclonal antibodies to HIV-1 envelope protein. Mice immunized with V1V2-SHB-SAPN raised V1V2 specific antibodies, that, most importantly, bound to gp70 V1V2, the correct conformational epitopes9. The SHB-SAPN core could have other functions beyond the role as a carrier for the HIV-1 V1V2 loop. Here we describe a detailed methodology for the expression, purification, refolding, and validation of the SHB-SAPN core. The sequence selection, nanoparticle design, the molecular cloning, and transformation of E. coli have been previously described9.

<table><tbody><tr><td>10x Tris/Glycine/SDS</td><td>BioRad</td><td>1610732</td><td>1 L</td></tr><tr><td>2-Mercaptoethanol</td><td>BioRad</td><td>1610710</td><td>25 mL</td></tr><tr><td>2-propanol</td><td>Fisher</td><td>BP26181</td><td>4 L</td></tr><tr><td>2x Laemmli Sample Buffer</td><td>BioRad</td><td>1610737</td><td>30 mL</td></tr><tr><td>40ul Cuvette Pack of 100 with Stoppers</td><td>Malvern Panalytical</td><td>ZEN0040</td><td>100 pack</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 &amp;micro;l</td><td>BioRad</td><td>4561093</td><td>10 pack</td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-25</td><td>25 g</td></tr><tr><td>Anti-6X His tag antibody [HIS.H8]</td><td>AbCam</td><td>ab18184</td><td>100 mg</td></tr><tr><td>Anti-HIV-1 gp41 Monoclonal (167-D IV)</td><td>AIDS Reagent Repository</td><td>11681</td><td>100 mg</td></tr><tr><td>BCIP/NBT Substrate, Solution</td><td>Southern Biotech</td><td>0302-01 &amp;nbsp;</td><td>100 mL</td></tr><tr><td>Corning&amp;nbsp;Disposable Vacuum Filter/Storage Systems</td><td>Fisher</td><td>09-761-108</td><td>A variety of sizes</td></tr><tr><td>Formvar/Carbon 400 mesh, Copper approx. grid hole size: 42&amp;micro;m</td><td>Ted Pella, Inc</td><td>01754-F</td><td>25 pack</td></tr><tr><td>GE Healthcare&amp;nbsp;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>Goat Anti-Mouse IgG H&amp;amp;L (Alkaline Phosphatase)</td><td>ABCam</td><td>ab97020</td><td>1 mg</td></tr><tr><td>Imidazole</td><td>Fisher</td><td>O3196-500</td><td>500 g</td></tr><tr><td>Instant NonFat Dry Milk</td><td>Quality Biological</td><td>A614-1003</td><td>10 pack</td></tr><tr><td>Kinetic-QCL Kinetic Chormogenic LAL Assay</td><td>Lonza Walkersville</td><td>50650U</td><td>192 Test Kit</td></tr><tr><td>LAL Reagent Grade Multi-well Plates</td><td>Lonza Walkersville</td><td>25-340</td><td>1 plate</td></tr><tr><td>Magic Media&lt;em&gt; E. coli&lt;/em&gt; 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>Mouse Anti-Human IgG Fc-AP</td><td>Southern Biotech</td><td>9040-04 &amp;nbsp;</td><td>1.0 mL</td></tr><tr><td>One Shot BL21 Star (DE3) Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>ThermoFisher</td><td>C601003</td><td>20 vials</td></tr><tr><td>Precision Plus Protein Unstained Protein Standards, Strep-tagged recombinant,</td><td>BioRad</td><td>1610363</td><td>1 mL</td></tr><tr><td>Slide-A-Lyzer Dialysis Cassettes, 10K MWCO, 12 mL</td><td>ThermoFisher</td><td>66810</td><td>8 pack</td></tr><tr><td>Sodium Chloride</td><td>Fisher</td><td>BP358-212</td><td>2.5 kg</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>Tris-(2-carboxyethyl)phosphine hydrochloride</td><td>Biosynth International</td><td>C-1818</td><td>100 g</td></tr><tr><td>Uranyl Acetate, Reagent, A.C.S</td><td>Electron Micoscopy Services</td><td>541-09-3</td><td>25 g</td></tr><tr><td>Urea</td><td>Fisher</td><td>BP169-500</td><td>2.5 kg</td></tr><tr><td>Whatman qualitative filter paper</td><td>Sigma Aldrich</td><td>WHA10010155</td><td>pack of 500</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&amp;nbsp;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ChromLab Software ver 4</td><td>BioRad</td><td>12009390</td><td>Software</td></tr><tr><td>Epoch 2 Microplate Spectrophotometer</td><td>BioTek</td><td>EPOCH2</td><td>Plate Reader</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>Gel Doc EZ Gel Documentation System</td><td>BioRad</td><td>1708270</td><td>Gel Imager for Stain free Gels</td></tr><tr><td>JEOL TEM</td><td>JEOL</td><td>1400</td><td>Transmission Electron Microscope</td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels</td><td>BioRad</td><td>1658004</td><td>To run gels</td></tr><tr><td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td><td>ThermoFisher</td><td>ND-ONE-W</td><td>For Protein Concentration</td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr><tr><td>NanoSight NTA software NTA</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr><tr><td>New Brunswick Innova 44/44R</td><td>Eppendorf</td><td>M1282-0000</td><td>Incubator/Shaker</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>PELCO easiGlow Glow Discharge Cleaning System</td><td>Ted Pella, INC</td><td>91000S</td><td>To clean grids</td></tr><tr><td>PowerPac Universal Power Supply</td><td>BioRad</td><td>1645070</td><td>To run gels</td></tr><tr><td>Rocker Shaker</td><td>Daigger</td><td>EF5536A</td><td>For Western</td></tr><tr><td>Sonifer 450</td><td>Branson</td><td>also known as 096-145075&amp;nbsp;</td><td>Sonicator</td></tr><tr><td>Thermo Scientific Sorvall LYNX 4000 Superspeed Centrifuge</td><td>ThermoFisher</td><td>75-006-580</td><td>Centrifuge</td></tr><tr><td>Trans-Blot Turbo Mini Nitrocellulose Transfer Packs</td><td>BioRad</td><td>1704158</td><td>For Western</td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BioRad</td><td>1704150</td><td>For Western</td></tr><tr><td>Vortex-Genie 2</td><td>Daigger</td><td>EF3030A</td><td>Vortex</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr><tr><td>Zetasizer Software</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr></tbody></table>

production of e. coli-expressed self-assembling protein nanoparticles for vaccines requiring trimeric epitope presentation

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

The fully assembled SHB-SAPN shown here is built upon protein sequences (Figure 1A) that are predicted to fold into a particle that contains 60 copies of the monomer (Figure 1B). Figure 2 provides an outline of the method for the production, purification, and identification of the SHB-SAPN core. E. coli from a glycerol stock that contained a pPep-T expression vector with gene sequence of the SHB-SAPN core were induced in BL21 (DE3) E. coli. Bacterial cells were successfully grown and lysed under denaturing and reducing conditions.
Total cell lysate was used to purify SHB-SAPN monomers by FPLC using a Ni2+ column (Figure 3A). The FPLC chromatograph demonstrates that protein eluted both at 150 mM and 500 mM imidazole (Figure 3A). The chromatogram also shows two other peaks at 185 mL and 210 mL total volume corresponding to the isopropanol wash and the imidazole-free wash, respectively. The fractions and the purity of the recombinant protein were identified by gradient SDS-PAGE gels (Figure 3B). The protein of interest was primarily located in fractions 68‒79 (278‒300 mL total volume). These fractions were combined for further analyses. Western blot with anti-His antibody (N-terminal) and 167-D-IV antibody (C-terminus) indicated that the pooled fractions were indeed the protein of interest (Figure 4A,B). These blots also demonstrated the presence of the SHB-SAPN multimers. Earlier washes and elution fractions tended to contain a higher concentration of multimerized protein and were therefore excluded.
The samples that contained the protein monomers of interest were folded into the fully assembled SHB-SAPN by dialysis. Particle size distribution was determined by DLS and nanoparticle tracking analysis (Figure 5A,B). The DLS identified particles with a Z-average hydrodynamic diameter of 67 nm while the NTA system measured a mean size of 81 nm. The slight size differences were due to the particle sizing techniques, however the size from both analyses were in the expected range of 20‒100 nm8,24,25. SHB-SAPNs were visualized by TEM and the images showed well-formed individual particles with the size distribution obtained from the two particle sizing techniques (Figure 5C).
During the purification of the protein, the column was washed with isopropanol to decrease the LPS contamination in the final SHB-SAPN product. To verify if the endotoxin level was acceptable for immunization, the concentration of LPS in SHB-SAPN samples purified with or without the isopropanol wash step was determined by a kinetic LAL assay. The results indicated that the isopropanol wash decreased the endotoxin levels from &gt;0.25 EU/µg to 0.010 EU/µg of SHB-SAPN protein (Table 1).
<img alt="Protein sequence and structure diagram; sequence alignment, molecular structure analysis, bioinformatics." class="xfigimg" src="/files/ftp_upload/60103/60103fig1.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: SHB-SAPN protein sequence and structure. (A) The amino acid sequence of the SHB-SAPN monomer. (B) Computer model of the structure of the fully assembled SHB-SAPN core consisting of 60 protein monomers. Color scheme for amino acid sequences: Grey = HisTag; Green = pentamer; Dark blue = de novo designed trimer; Light blue = six-helix bundle. <a href="https://www.jove.com/files/ftp_upload/60103/60103fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial protein purification process flowchart with SDS-PAGE, Western blot, TEM visualization steps." class="xfigimg" src="/files/ftp_upload/60103/60103fig2.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: Flowchart of the protocol for SHB-SAPN production. Color scheme: Black = expression of the protein monomer in E. coli; Dark grey = monomer purification; Medium grey = monomer identification; Light grey = refolding and characterization; White = fully assembled SHB-SAPN product. In steps labeled with dark and medium grey, the protein is under denaturing and reducing conditions. <a href="https://www.jove.com/files/ftp_upload/60103/60103fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography protein purification: Elution profile &amp; SDS-PAGE, fractions E150, E500 analysis." class="xfigimg" src="/files/ftp_upload/60103/60103fig3.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3: Protein purification of the SHB-SAPN monomers. (A) Chromatograph from the FPLC purification. Green line above the chromatogram indicates the purification steps. Blue line in the chromatogram represents the optical density of the fractions at 280 nm wavelength. The black line shows what percent of buffer B (8 M urea, 20 mM Tris, 50 mM sodium phosphate monobasic, 5 mM TCEP, 500 mM Imidazole, pH 8.5) that was used in each stage of the purification. (B) SDS-PAGE gels of the pooled fractions from the lysate (L), flow through (FT), first wash (W1), isopropanol wash (W2), third wash (W3), and individual fractions from the 150 mM imidazole (E150) and 500 mM imidazole (E500) elution steps of the purification. Molecular markers in the first lane (M) identify bands between 10 and 250 kDa. Target protein is indicated by a black arrow. <a href="https://www.jove.com/files/ftp_upload/60103/60103fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SDS-PAGE electrophoresis results; protein bands with molecular weight markers, indicating separation." class="xfigimg" src="/files/ftp_upload/60103/60103fig4.jpg" data-altupdatedat="1788282455000" data-alt="Figure 4"> 
Figure 4: Identification of the protein by western blot. All lanes are loaded with 100 ng of protein. (A) Results of a western blot with anti-6x HisTag. (B) Results of a western blot with a 167-D-IV HIV-1 monoclonal antibody. Lanes are labeled as: M = molecular weight marker; 1 = lysate; 2 = Flow through; 3 = first wash; 4 = isopropanol wash (second wash); 5 = third wash; 6 = pooled volume fractions 56‒61 (first elution peak), 7 = pooled volume factions 62‒67 (between the two peaks), 8 = pooled volume fractions 68‒78 (second elution peak). Target protein with the expected band size of 18.07 kDa as the monomeric SHB-SAPN band is indicated by a black arrow. Extra banding in lanes 7 and 8 are dimers, trimers, and multimers of the SHB-SAPN (red arrow). <a href="https://www.jove.com/files/ftp_upload/60103/60103fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Nanoparticle size distribution graph, intensity vs. diameter plot, and electron microscope image." class="xfigimg" src="/files/ftp_upload/60103/60103fig5v2.jpg" data-altupdatedat="1788282455000" data-alt="Figure 5"> 
Figure 5: Characterization of the refolded SHB-SAPN. (A). Particle size distribution as determined by DLS. (B) Particle size as determined by nanoparticle tracking (system). (C) Visualization of the SHB-SAPN particles by TEM. <a href="https://www.jove.com/files/ftp_upload/60103/60103fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Endotoxin (EU/mL)</td>
			<td>Endotoxin (EU/µg of Protein)</td>
		</tr>
		<tr>
			<td>SAPN with Isopropanol Wash</td>
			<td>2.02</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>SAPN without Isopropanol Wash</td>
			<td>&gt;50</td>
			<td>&gt;0.25</td>
		</tr>
		<tr>
			<td>Negative Control</td>
			<td>Below detection level</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>
Table 1: Endotoxin levels of refolded SHB-SAPNs. Endotoxin levels in SHB-SAPN samples purified with or without an isopropanol wash presented both as endotoxin units/mL and endotoxin units/µg of SHB-SAPN protein.

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

production-e-coli-expressed-self-assembling-protein-nanoparticles-for

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