Abstract:Ferritin, with the primary function of iron storage, is a nearly ubiquitous protein found in most living organisms. Our recent investigations suggest that ferritin can assemble nanoparticles. So we use ferritin as a novel type of delivery vehicle for recombinant epitope vaccines. And, we found that ferritin form nonnative aggregates depended sensitively on NaCl concentrations. Here, we report that ferritin is an ion-sensitive protein and has the attribute of salt-dependent aggregation. Our results indicate tha… Show more
“…It has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins ( 53 , 54 ). We examined the effect of salt concentration on the formation and stability of the RBD-[SSG]-FR NPs at various concentrations (0–300 mM).…”
Section: Resultsmentioning
confidence: 99%
“…As intrinsic factors, the concentration of salts and Fe 2+ also influenced the assembly and stability of NPs (Figures 3 and 4 ). The ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of NaCl ( 54 ). The assembly of the hybrid MERS-CoV NPs revealed an interesting change in salt dependence, with 200–225 mM NaCl buffer as optimal condition as confirmed by EM and DLS analyses (Figure 3 ).…”
Section: Discussionmentioning
confidence: 99%
“…The assembly of the hybrid MERS-CoV NPs revealed an interesting change in salt dependence, with 200–225 mM NaCl buffer as optimal condition as confirmed by EM and DLS analyses (Figure 3 ). The change in salt dependence was probably due to the presence of electrostatic interactions among RBD domains ( 54 , 67 ). The dependence on Fe 2+ was not surprising considering that ferritin has an intrinsic ability to interact with Fe 2+ to form ferritin-iron cores ( 55 ).…”
The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe2+, salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections.
“…It has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins ( 53 , 54 ). We examined the effect of salt concentration on the formation and stability of the RBD-[SSG]-FR NPs at various concentrations (0–300 mM).…”
Section: Resultsmentioning
confidence: 99%
“…As intrinsic factors, the concentration of salts and Fe 2+ also influenced the assembly and stability of NPs (Figures 3 and 4 ). The ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of NaCl ( 54 ). The assembly of the hybrid MERS-CoV NPs revealed an interesting change in salt dependence, with 200–225 mM NaCl buffer as optimal condition as confirmed by EM and DLS analyses (Figure 3 ).…”
Section: Discussionmentioning
confidence: 99%
“…The assembly of the hybrid MERS-CoV NPs revealed an interesting change in salt dependence, with 200–225 mM NaCl buffer as optimal condition as confirmed by EM and DLS analyses (Figure 3 ). The change in salt dependence was probably due to the presence of electrostatic interactions among RBD domains ( 54 , 67 ). The dependence on Fe 2+ was not surprising considering that ferritin has an intrinsic ability to interact with Fe 2+ to form ferritin-iron cores ( 55 ).…”
The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe2+, salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections.
“…To demonstrate tES rescue of GFPuv expression, GFPuv was induced at 0.8 (O.D. 600 nm) due to its extremely low expression in late exponential phase 10 . While leaving isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration constant (0.4 mM), L-arabinose was varied from 0.001 to 0.1%.…”
Section: Resultsmentioning
confidence: 99%
“…1a ) 8 , 9 . Second, while eukaryotic ferritin assemblies are stable in low-salt concentrations 10 , AfFtn disassociates in low-salt (NaCl) concentrations 8 . Fig.…”
The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.
Nanoparticles are used as carriers for the delivery of drugs and imaging agents.Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g −1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5′-untranslated regions in PCPs, and selected the bestperforming construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg −1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for
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