Recombinant Ferritin Research Articles

Influenza Nanovaccines: Intranasal Nanovaccine Confers Homo‐ and Hetero‐Subtypic Influenza Protection (Small 13/2018)

In article number 1703207, Jianjun Chen, Zongqiang Cui, and co-workers develop an intranasal nanovaccine which provides protection against homo- and heterosubtypic influenza. The 3M 2e-rHF nanovaccine is constructed by displaying three-sequential repeats of the influenza conserved antigen M2e on the self-assembling recombinant human heavy chain ferritin (rHF) cage. Intranasal vaccination with 3M2e-rHF in the absence of adjuvants confers complete protection against a lethal infection of homosubtypic H1N1 and heterosubtypic H9N2 virus.

Intranasal Nanovaccine Confers Homo- and Hetero-Subtypic Influenza Protection.

Cross-protective and non-invasively administered vaccines are attractive and highly desired for the control of influenza. Self-assembling nanotechnology provides an opportunity for the development of vaccines with superior performance. In this study, an intranasal nanovaccine is developed targeting the conserved ectodomain of influenza matrix protein 2(M2e). 3-sequential repeats of M2e (3M2e) is presented on the self-assembling recombinant human heavy chain ferritin (rHF) cage to form the 3M2e-rHF nanoparticle. Intranasal vaccination with 3M2e-rHF nanoparticles in the absence of an adjuvant induces robust immune responses, including high titers of sera M2e-specific IgG antibodies, T-cell immune responses, and mucosal secretory-IgA antibodies in mice. The 3M2e-rHF nanoparticles also confer complete protection against a lethal infection of homo-subtypic H1N1 and hetero-subtypic H9N2 virus. An analysis of the mechanism of protection underlying the intranasal immunization with the 3M2e-rHF nanoparticle indicates that M2e-specific mucosal secretory-IgA and T-cell immune responses may play critical roles in the prevention of infection. The results suggest that the 3M2e-rHF nanoparticle is a promising, needle-free, intranasally administered, cross-protective influenza vaccine. The use of self-assembling nanovaccines could be an ideal strategy for developing vaccines with characteristics such as high immunogenicity, cross-protection, and convenient administration, as well as being economical and suitable for large-scale production.

First biochemical and crystallographic characterization of a fast-performing ferritin from a marine invertebrate.

Ferritin, a multimeric cage-like enzyme, is integral to iron metabolism across all phyla through the sequestration and storage of iron through efficient ferroxidase activity. While ferritin sequences from ∼900 species have been identified, crystal structures from only 50 species have been reported, the majority from bacterial origin. We recently isolated a secreted ferritin from the marine invertebrate Chaetopterus sp. (parchment tube worm), which resides in muddy coastal seafloors. Here, we present the first ferritin from a marine invertebrate to be crystallized and its biochemical characterization. The initial ferroxidase reaction rate of recombinant Chaetopterus ferritin (ChF) is 8-fold faster than that of recombinant human heavy-chain ferritin (HuHF). To our knowledge, this protein exhibits the fastest catalytic performance ever described for a ferritin variant. In addition to the high-velocity ferroxidase activity, ChF is unique in that it is secreted by Chaetopterus in a bioluminescent mucus. Previous work has linked the availability of Fe2+ to this long-lived bioluminescence, suggesting a potential function for the secreted ferritin. Comparative biochemical analyses indicated that both ChF and HuHF showed similar behavior toward changes in pH, temperature, and salt concentration. Comparison of their crystal structures shows no significant differences in the catalytic sites. Notable differences were found in the residues that line both 3-fold and 4-fold pores, potentially leading to increased flexibility, reduced steric hindrance, or a more efficient pathway for Fe2+ transportation to the ferroxidase site. These suggested residues could contribute to the understanding of iron translocation through the ferritin shell to the ferroxidase site.

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.

Novel recombinant ferritin from Perinereis sp. alters iron profiles and improves the performance of weaned pigs

The current study evaluated the effect of dietary recombinant ferritin (FER) from Perinereis sp. expressed in E. coli as an intracellular protein and Bacillus subtilis as an extracellular protein on serum profiles, growth performance, apparent total tract digestibility (ATTD), diarrhea scores and fecal microbiota in weanling pigs. A total of 160 weanling pigs [(Yorkshire×Landrace)×Duroc] with an average BW of (6.62±0.33kg; 21 d of age) were used in a 42 d study. Piglets were randomly allotted to 4 experimental diets with 8 replicate pens and 5 pigs per pen. Dietary treatments were as follows: CON, corn-soybean meal basal diet; FER05, basal diet+5g/tonne FER; FER10, basal diet+10g/tonne FER; FER15, basal diet+15g/tonne FER. Pigs were fed with phase 1 (1–14 d) and phase 2 (15–42 d) diets in the form of mash. Average daily feed intake (ADFI) was linearly increased (P=0.027) by FER15 supplementation compared with CON diet during d 15–28. Piglets fed FER10 diet had increased average daily gain (ADG) and growth efficiency (G:F) compared with the CON diet during d 29–42 (P=0.019 and 0.010, respectively) and overall (P=0.012 and 0.002, respectively). Moreover, G:F was linearly increased by FER supplementation during d 1–42 (P<0.001). Apparent dry matter (DM) digestibility was higher (linear, P=0.030) on the FER15 treatment compared to the CON on d 14. On d 14 dietary supplementation of FER linearly increased iron (Fe), ferritin and hemoglobin (Hb) concentration (P=0.002,<0.001 and 0.015, respectively). Pigs fed FER diets also had higher Fe and total iron-binding capacity (TIBC) concentrations at d 42 compared with those fed CON diet (linear, P<0.001 and 0.038). The diarrhea score and fecal microbiota were not affected by dietary supplementation with FER during the whole experiment (P>0.10). In conclusion, results showed that FER improved serum Fe, TIBC, fettitin and Hb and that these changes were associated with improved growth performance in weanling piglets.

Efficient expression of recombinant human heavy chain ferritin (FTH1) with modified peptides

Human heavy chain ferritin (FTH1) can self-assemble into a diameter of 12-nm spherical cage with an interior cavity of 8 nm in diameter. FTH1 has great potential as a nanocage in molecular imaging and drug delivery. Different peptides have been fused with FTH1 for targeting delivery; however, the expression of FTH1 modified with peptides in soluble form is not equivalent to natural FTH1. As shown in recent study, a novel scaffold protein --thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx)--exhibits a superior solubilization capacity and thermal stability [19]. Here we report a new construct (FTH1-PfTrx-His) that can be easily expressed and purified in Escherichia coli. Of note, different peptides inserted into FTH1-PfTrx-His did not influence the expression of proteins. Finally, the doxorubicin packaging ability of FTH1-PfTrx-His is comparable to natural FTH1. Our results showed that FTH1-PfTrx-His had a potential role as a novel peptide-modified ferritin carrier for drugs or imaging probes.

"Silent" Amino Acid Residues at Key Subunit Interfaces Regulate the Geometry of Protein Nanocages.

Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy-key subunit interface redesign (KSIR)-that alters a natural subunit-subunit interface by selective deletion of a small number of "silent" amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

Expression, purification, and characterization of recombinant human H-chain ferritin

ABSTRACTBased on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15 mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.

고초균에서 흰이빨참갯지렁이 페리틴 단백질의 분비 및 사료 효율성

Ferritin is known to regulate iron metabolism and maintain iron in a variety of the eukaryotic organisms. The region encoding the mature ferritin (0.47 kb, H-type) of Periserrula leucophryna was amplified using the designed primers including restriction enzyme site and termination codon and subcloned in frame to the pRBAS secretion vector containing the signal sequence, RBS, and promoter of amylase gene (E. coli-Bacillus shuttle vector), resulting in recombinant pRBAS-PLF vector. Recombinant ferritin (18 kDa) was correctly processed and secreted from Bacillus subtilis LKS strain harboring the pRBAS-PLF vector and quantitatively analyzed by SDS-PAGE and western blot, respectively. Secretion of the ferritin was optimized by culture conditions (host, medium, temperature, nitrogen source) in 3 L batch culture and 5 L jar fermenter. Finally. the ferritin was largely produced using 50 L fermenter as the following conditions; at <TEX>$30^{\circ}C$</TEX>, 150 rpm, 1 vvm in Bacillus subtilis LKS using PY medium. The secreted ferritin was maximally measured (approximately 177.6 ug/ml) when the cell density reached to 14.4 at <TEX>$OD_{600}$</TEX> (20 h incubation). The iron binding activity was confirmed by Perls' staining in 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in the culture broth after secretion. Biologically, the culture broth and powder type containing ferritin were tested for possibility as feed additive in chicken broiler. As a result, the ferritin stimulated the growth of chick broil and improved feed efficiency and production index.

Insights on the (Auto)Photocatalysis of Ferritin.

Traditionally, ferritin has been considered a photocatalyst capable of photo-oxidizing organic molecules and transferring electrons to external electron acceptors when irradiated by UV-visible light. We have designed new approaches to resolve the uncertainties regarding its photocatalytical mechanism. Experiments with an Fe(II) chelator, an electrochromic indicator, and recombinant ferritin proteins indicate that the excited electrons at the conduction band of the ferritin core do not cross the protein shell. Instead, irradiation causes the electrons to reduce the ferrihydrite core to produce Fe(II) ions. These Fe(II) ions exit the protein shell to reduce electron acceptors. In the absence of electron acceptors or chelators, Fe(II) re-enters ferritin.

Energetics of surface confined ferritin during iron loading

We report on the first quantitative picture on how iron loading inside ferritin molecules occurs when they are self-assembled onto solid surfaces. Recombinant human ferritin H-chain with ferroxidase activity was adsorbed onto microcantilever beams to form a stable close-packed thin film. The obtained nanomechanical system was used to track in real time the energetics of inter-ferritin surface interactions during incubation with Fe(II) for iron loading. We observed that iron loading is accompanied by increasing attractive in-plane inter-ferritin interactions able to perform a maximum surface work of 6.0±1.5mJ/m2, corresponding to a surface energy variation per ferritin of about 40kbT. Unique to this protein surface transformation, part of the surface work is exerted by the attractive electrostatic forces arising among the new born nanosized iron cores inside the ferritin shells. The remaining work comes from subtle action of steric, bridging and depletion forces. These findings are of fundamental interest and add important information for the rational development of ferritin nanotechnology.

Salt-Dependent Aggregation and Assembly of E coli-Expressed Ferritin

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 that recombinant ferritin can be released as a soluble form from Escherichia coli at low NaCl concentrations (≤50 mmol/L). Moreover, this result affords us to confirm a proper self-assembling solution for soluble ferritin or other ferritin-based fusion proteins to assemble nanoparticles.

Expression, purification, and characterization of recombinant human L-chain ferritin

Ferritins form nanocage architectures and demonstrate their potential to serve as functional nanomaterials with potential applications in medical imaging and therapy. In our study, the cDNA of human L-chain ferritin was cloned into plasmid pET-28a for its overexpression in Escherichia coli. However, the recombinant human L-chain ferritin (rLF) was prone to form inclusion bodies. Molecular chaperones were co-expressed with rLF to facilitate its correct folding. Our results showed that the solubility of rLF was increased about 3-fold in the presence of molecular chaperones, including GroEL, GroES and trigger factor. Taking advantage of its N-terminal His-tag, rLF was then purified with Ni-affinity chromatography. With a yield of 10 mg/L from bacterial culture, the purified rLF was analyzed by circular dichroism spectrometry for its secondary structure. Furthermore, the rLF nanocages were characterized using dynamic light scattering and transmission electron microscopy.

Preparation and representation of recombinant Mn-ferritin flower-like spherical aggregates from marine invertebrates.

Ferritin has important functions in the transition and storage of toxic metal ions, but its regulation and function in many invertebrate species are still largely unknown. In our previous work, the cDNA sequence of Sinonovacula constricta, Apostichopus japonicas and Acaudina leucoprocta were constructed and efficiently expressed in E. Coli BL21 under IPTG induction. In this follow-up study, the recombinant ferritins were exposed to heavy metal manganese. The manganese concentration levels in three recombinant ferritins were greater than horse spleen ferritin (HSF). Compared with HSF, the amount of manganese enrichment in the three recombinant ferritins was 1.75-fold, 3.25-fold and 2.42-fold increases in ScFER, AjFER, and AlFER, respectively. After phosphate stimulation, the concentration of manganese increased and was higher than the ordinary dialysis control groups. The ScFER was four times its baseline value. The AjFER and AlFER were 1.4- and 8-fold higher, respectively. The AlFER sample stimulated by phosphate was 22-fold that of HSF. The morphologies of the resulting Mn-Ferritin from different marine invertebrates were characterized with scanning electron microscopy. Surface morphologies were lamella flower-like and are consistent with changes in surface morphologies of the standard Mn-HSF. Invertebrate recombinant ferritin and HSF both can uptake manganese. We found that the structure of A. leucoproctarecombinant Mn-Ferritin aggregate changed over time. The surface formed lamella flower-like aggregate, but gradually merged to create a relatively uniform plate-like phase of aggregate spherically and fused without clear boundaries.

Different protein of Echinococcus granulosus stimulates dendritic induced immune response.

Cystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.

Ferritin protect shrimp Litopenaeus vannamei from WSSV infection by inhibiting virus replication

Iron is considered as an essential element for all living organisms. Therefore, limiting iron availability may be key part of the host's innate immune response to various pathogens. Ferritin is a major iron storage protein in living cells and plays an important role in iron homeostasis. One way the host can transiently reduce iron bioavailability is by ferritin over expression. In invertebrates, ferritin was found to be up-regulated after pathogens challenge and is considered to be an important element in the innate immune system. This study was designed to investigate the involvement of ferritin in shrimp Litopenaeus vannamei defense against WSSV. We discovered that the viral load of shrimp injected with recombinant ferritin protein was lower than that of control group. The suppression of ferritin by dsRNA increased susceptibility to WSSV with 3-fold high viral copies. The present study documented that ferritin protected shrimp L. vannamei from WSSV by inhibiting virus replication. We presume that ferritin reduce iron availability, leading to inhibit the activity of ribonucleotide reductase and delay the replication of virus genome. This study provided new insights into the understanding of molecular responses and defense mechanisms in shrimp against WSSV.

The crystal structure of ferritin from Chlorobium tepidum reveals a new conformation of the 4-fold channel for this protein family.

Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3Å; corresponding to a diferric μ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity.

Identification and characterization of a Macrobrachium nipponense ferritin subunit regulated by iron ion and pathogen challenge

Ferritin, a major iron storage protein in most living organisms, plays a crucial role in iron metabolism. In this study, the ferritin subunit MnFer was identified in the oriental river prawn (Macrobrachium nipponense) and functionally characterized. The full-length cDNA of MnFer is 999 bp in size with a 122-bp 5′-untranslated region (UTR), a 364-bp 3′-UTR and a 513-bp open reading frame that encodes a protein possessing 171 amino acids and a deduced molecular weight of 19.40 kDa. Prawn ferritin transcripts are expressed in muscle, heart, hepatopancreas, gill, hemocytes, ovary and testis. Quantitative real-time PCR revealed that the abundance of ferritin transcript was highest in the hepatopancreas followed by muscle. Ferritin transcript expression in muscle increased six-fold 3 h after the injection of iron. In the gill, a four-fold increase in ferritin transcript expression was detected 3 h post-injection; the expression remained elevated for 48 h. Heart ferritin mRNA expression increased up to seven-fold at 24 h post-injection. No significant difference was found in the hepatopancreas. The iron binding capacity of recombinant ferritin protein was also demonstrated in this study. In hemocyte experiments, the transcriptional expression of MnFer showed the strongest response to Aeromonas hydrophila. As a whole, our study suggested that the ferritin from M. nipponense may play critical roles in cellular and organismic iron homeostasis along with in innate immune defense.

Encapsulation of curcumin in recombinant human H-chain ferritin increases its water-solubility and stability

Curcumin is a natural bioactive agent found in turmeric (Curcuma longa) and has many health-promoting benefits. However, its poor water-solubility and thermal and photostability limit its application in foodstuffs. In the present study, recombinant human H-chain ferritin (rHuHF), a typical shell-like protein with an inner diameter as ~8nm was used to encapsulate curcumin by taking advantage of the reversible dissociation and reassembly characteristic of apoferritin at different pH values. We demonstrated that curcumin molecules were successfully encapsulated within protein cages with a curcumin/protein molar ratio of 14.7 to 1. Upon such molecular encapsulation, curcumin molecules become greatly water-soluble, while their thermal and photostability were greatly improved due to protection by protein cages as compared to free analogs. This novel approach reported in this study has a potential to be applied in other poorly water-soluble bioactive compounds.

Identification and functional characterization of a novel ferritin subunit from the tropical sea cucumber, Stichopus monotuberculatus

Ferritin is one of the major non-harm iron storage proteins that found in most cell types of animals, plants and microorganisms. In this study, a ferritin subunit named StmFer was identified from the sea cucumber (Stichopus monotuberculatus) and characterized functionally. The full-length cDNA of StmFer is 1184 bp in size with a 5′-untranslated region (UTR) of 131 bp, a 3′-UTR of 531 bp and an open reading frame of 522 bp that encoding a protein of 173 amino acids with a deduced molecular weight of 19.95 kDa. StmFer possesses both the ferroxidase center of vertebrate ferritin heavy subunit and iron nucleation sites of vertebrate ferritin light subunit. For the gene structure, StmFer contains only three exons separated by two introns. Higher levels of mRNA expression were noticed in intestine and coelomocyte of S. monotuberculatus by northern blot analysis. In in vitro experiments performed in coelomocytes, transcriptional expression of StmFer showed the strongest response to polyriboinosinic polyribocytidylic acid [Poly (I:C)] (9.08 fold up-regulation), followed by lipopolysaccharides (LPS), ferrous chloride (FeCl2) and inactivated bacteria (Vibrio alginolyticus) (7.84, 7.41 and 4.90 fold up-regulation, respectively) after 3 h post-challenge. In addition, the anti-oxidation activity and iron binding capacity of recombinant ferritin protein were demonstrated in this study. As a whole, our study suggested that the ferritin from sea cucumber may play critical roles not only in the cellular and organismic iron homeostasis, but also in the innate immune defense.