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.

<ol>
	<li>Karch, C. P., Burkhard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccine+technologies:+From+whole+organisms+to+rationally+designed+protein+assemblies.">Vaccine technologies: From whole organisms to rationally designed protein assemblies.</a> Biochemical Pharmacology. 120, 1-14 (2016).</li><li>Snapper, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Immunologic+Properties+of+Soluble+Versus+Particulate+Antigens.">Distinct Immunologic Properties of Soluble Versus Particulate Antigens.</a> Frontiers in Immunology. 9, 598 (2018).</li><li>Kelly, H. G., Kent, S. J., Wheatley, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+basis+for+enhanced+immunity+of+nanoparticle+vaccines.">Immunological basis for enhanced immunity of nanoparticle vaccines.</a> Expert Review of Vaccines. , 1-12 (2019).</li><li>Yeates, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+Principles+for+Designing+Highly+Symmetric+Self-Assembling+Protein+Nanomaterials.">Geometric Principles for Designing Highly Symmetric Self-Assembling Protein Nanomaterials.</a> Annual Review of Biophysics. 46, 23-42 (2017).</li><li>Marcandalli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Potent+Neutralizing+Antibody+Responses+by+a+Designed+Protein+Nanoparticle+Vaccine+for+Respiratory+Syncytial+Virus.">Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.</a> Cell. 176 (6), 1420-1431 (2019).</li><li>Ross, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decorating+Self-Assembled+Peptide+Cages+with+Proteins.">Decorating Self-Assembled Peptide Cages with Proteins.</a> ACS Nano. 11 (8), 7901-7914 (2017).</li><li>Karch, C. P., Matyas, G. R., Burkhard, P., Beck, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembling+Protein+Nanoparticles:+implications+for+HIV-1+vaccine+development.">Self-Assembling Protein Nanoparticles: implications for HIV-1 vaccine development.</a> Nanomedicine (Lond). 13 (17), 2121-2125 (2018).</li><li>Raman, S., Machaidze, G., Lustig, A., Aebi, U., Burkhard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+design+of+peptides+that+self-assemble+into+regular+polyhedral+nanoparticles).">Structure-based design of peptides that self-assemble into regular polyhedral nanoparticles).</a> Nanomedicine. 2 (2), 95-102 (2006).</li><li>Karch, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+characterization+of+a+self-assembling+protein+nanoparticle+displaying+HIV-1+Env+V1V2+loop+in+a+native-like+trimeric+conformation+as+vaccine+antigen.">Design and characterization of a self-assembling protein nanoparticle displaying HIV-1 Env V1V2 loop in a native-like trimeric conformation as vaccine antigen.</a> Nanomedicine. , (2018).</li><li>Karch, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+P.+falciparum+specific+coiled-coil+domain+to+construct+a+self-assembling+protein+nanoparticle+vaccine+to+prevent+malaria.">The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.</a> Journal of Nanobiotechnology. 15 (1), 62 (2017).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-adjuvanted+nanoparticle+based+vaccine+against+infectious+bronchitis+virus.">A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus.</a> PLoS One. 13 (9), e0203771 (2018).</li><li>Wahome, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation-specific+display+of+4E10+and+2F5+epitopes+on+self-assembling+protein+nanoparticles+as+a+potential+HIV+vaccine.">Conformation-specific display of 4E10 and 2F5 epitopes on self-assembling protein nanoparticles as a potential HIV vaccine.</a> Chemical Biology &amp; Drug Design. 80 (3), 349-357 (2012).</li><li>El Bissati, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+a+novel+immunogenic+nanoparticle+platform+for+Toxoplasma+peptide+vaccine+in+HLA+transgenic+mice.">Effectiveness of a novel immunogenic nanoparticle platform for Toxoplasma peptide vaccine in HLA transgenic mice.</a> Vaccine. 32 (26), 3243-3248 (2014).</li><li>Kaba, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonadjuvanted+polypeptide+nanoparticle+vaccine+confers+long-lasting+protection+against+rodent+malaria.">A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria.</a> Journal of Immunology. 183 (11), 7268-7277 (2009).</li><li>Kaba, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibody+and+CD8++T-cell+responses+to+the+Plasmodium+falciparum+circumsporozoite+protein+induced+by+a+nanoparticle+vaccine.">Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine.</a> PLoS One. 7 (10), e48304 (2012).</li><li>Seth, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+self-assembling+protein+nanoparticle+vaccine+targeting+Plasmodium+falciparum+Circumsporozoite+Protein+delivered+in+three+Army+Liposome+Formulation+adjuvants.">Development of a self-assembling protein nanoparticle vaccine targeting Plasmodium falciparum Circumsporozoite Protein delivered in three Army Liposome Formulation adjuvants.</a> Vaccine. 35 (41), 5448-5454 (2017).</li><li>Kaba, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+protein+nanoparticles+with+built-in+flagellin+domains+increases+protective+efficacy+of+a+Plasmodium+falciparum+based+vaccine.">Self-assembling protein nanoparticles with built-in flagellin domains increases protective efficacy of a Plasmodium falciparum based vaccine.</a> Vaccine. 36 (6), 906-914 (2018).</li><li>El Bissati, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+nanovaccine+confers+robust+immunity+against+Toxoplasma.">Protein nanovaccine confers robust immunity against Toxoplasma.</a> NPJ Vaccines. 2, 24 (2017).</li><li>Karch, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+with+self-adjuvanted+protein+nanoparticles+provides+protection+against+lethal+influenza+challenge.">Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge.</a> Nanomedicine. 13 (1), 241-251 (2017).</li><li>Haynes, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-correlates+analysis+of+an+HIV-1+vaccine+efficacy+trial.">Immune-correlates analysis of an HIV-1 vaccine efficacy trial.</a> New England Journal of Medicine. 366 (14), 1275-1286 (2012).</li><li>Rerks-Ngarm, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+with+ALVAC+and+AIDSVAX+to+prevent+HIV-1+infection+in+Thailand.">Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand.</a> New England Journal of Medicine. 361 (23), 2209-2220 (2009).</li><li>O&#39; Connell, R. J., Kim, J. H., Excler, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV-1+gp120+V1V2+loop:+structure,+function+and+importance+for+vaccine+development.">The HIV-1 gp120 V1V2 loop: structure, function and importance for vaccine development.</a> Expert Review of Vaccines. 13 (12), 1489-1500 (2014).</li><li>Babapoor, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Vaccine+Using+Nanoparticle+Platform+to+Present+Immunogenic+M2e+against+Avian+Influenza+Infection.">A Novel Vaccine Using Nanoparticle Platform to Present Immunogenic M2e against Avian Influenza Infection.</a> Influenza Research and Treatment. 2011, 126794 (2011).</li><li>Indelicato, G., Burkhard, P., Twarock, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+self-assembling+protein+nanoparticle+architectures+for+applications+in+vaccine+design.">Classification of self-assembling protein nanoparticle architectures for applications in vaccine design.</a> Royal Society Open Science. 4 (4), 161092 (2017).</li><li>Indelicato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+Governing+the+Self-Assembly+of+Coiled-Coil+Protein+Nanoparticles.">Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles.</a> Biophysical Journal. 110 (3), 646-660 (2016).</li><li>Doll, T. A., Raman, S., Dey, R., Burkhard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+assemblies+and+their+biomedical+applications.">Nanoscale assemblies and their biomedical applications.</a> Journal of the Royal Society Interface. 10 (80), 20120740 (2013).</li><li>Rey, F. A., Lok, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+Features+of+Enveloped+Viruses+and+Implications+for+Immunogen+Design+for+Next-Generation+Vaccines.">Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines.</a> Cell. 172 (6), 1319-1334 (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Endotoxins.">Bacterial Endotoxins.</a> United States Pharmacopeia (USP). , (2011).</li><li>. Points to Consider (PTC) in the Characterization of Cell Lines Used to Produce Biologicals Available from: <a target="_blank" href="https://www.fda.gov/media/76255/download">https://www.fda.gov/media/76255/download</a> (1993)</li></ol>

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&#8211;20% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 &#181;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 &#160;</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Corning&#160;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&#181;m</td>
			<td>Ted Pella, Inc</td>
			<td>01754-F</td>
			<td>25 pack</td>
		</tr>
		<tr>
			<td>GE Healthcare&#160;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&#38;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 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>Mouse Anti-Human IgG Fc-AP</td>
			<td>Southern Biotech</td>
			<td>9040-04 &#160;</td>
			<td>1.0 mL</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>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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&#160;Equipment</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&#160;</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

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&#8210;79 (278&#8210;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&#8210;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 &#62;0.25 EU/&#181;g to 0.010 EU/&#181;g of SHB-SAPN protein (Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60103/60103fig1.jpg" /> 
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="Figure 2" class="xfigimg" src="/files/ftp_upload/60103/60103fig2.jpg" /> 
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="Figure 3" class="xfigimg" src="/files/ftp_upload/60103/60103fig3.jpg" /> 
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="Figure 4" class="xfigimg" src="/files/ftp_upload/60103/60103fig4.jpg" /> 
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&#8210;61 (first elution peak), 7 = pooled volume factions 62&#8210;67 (between the two peaks), 8 = pooled volume fractions 68&#8210;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="Figure 5" class="xfigimg" src="/files/ftp_upload/60103/60103fig5v2.jpg" /> 
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/&#181;g 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>&#62;50</td>
			<td>&#62;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/&#181;g of SHB-SAPN protein.

Watch this Scientific Journal Video about Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation at JoVE.com

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

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

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 ...

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

Research

JoVE Journal

Bioengineering

7.4K Views.  Walter Reed Army Institute of Research. A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

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

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...