• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.697-700
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• 2001

Iron is an essential nutrient for most organisms, which supplied to them in a protein-iron complex known as ferritin. Ferritins are multimeric proteins found in prokaryotes, plants and animals. They are consisted of spherical shell of 24 subunits defining a cavity of about 8nm in diameter, where an iron core is laid down. Expression of ferritin in recombinant E. coli at $37^{\circ}C$ led to the accumulation of recombinant ferritin. Insoluble form of ferritin was separated from disrupted cells, followed by various primary separation steps with two kinds of buffers. Collected samples from the primary steps were purified by DEAE-cellulose gels packed in a column. The fractions from the DEAE column were assayed to gain the amount and the purity of ferritin by using HPLC and SDS-PAGE.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.688-691
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• 2000

Ferritin, an iron-storage protein, is found in bacteria and some animal tissues such as liver, spleen, and bone marrow. It is more effective and causes less side reactions than the traditional ferrous sulfate, and thus has been used primarily to treat iron deficiency in pregnancy anaemia. Currently, the ferritin extracted from the bovine and equine spleens are sold as a commercial product. Its market is estimated as several hundreds of million US dollars. However, because of the recent warnings against the viral diseases of animal origins such as mad cow disease, a safer ferritin is sought after. Our research team has successfully developed a production process for recombinant human ferritin. Its expression titer from yeast is high enough to be economically viable, and its particle formation characteristics are as effective as well.

• Korean Journal of Veterinary Research
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• v.52 no.4
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• pp.263-268
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• 2012

In this study, we produced iron-fortified yeast (Saccharomyces cerevisiae) producing Sus scrofa ferritin heavy-chain to provide iron supplementation in anemic piglets. We determined whether iron-ferritin accumulated in recombinant yeasts could improve iron deficiency in mice. C57BL/6 male mice exposed to Fe-deficient diet for 2 weeks were given a single dose of ferrous ammonium sulfate (FAS), ferritin-producing recombinant yeast (APO), or APO reacted with iron ($Fe^{2+}$) (FER). The bioavailability of recombinant yeasts was examined by measuring body weight gain, hemoglobin concentration and hematocrit value 1 week later. In addition, ferritin protein levels were evaluated by western blot analysis and iron stores in tissues were measured by inductively coupled plasma spectrometer. We found that anemic mice treated with FER exhibited increased levels of ferritin heavy-chain in spleen and liver. Consistently, this treatment restored the iron concentration in these tissues. In addition, this treatment significantly increased hemoglobin value and the hematocrit ratio. Furthermore, FER treatment significantly enhanced body weight gain. These results suggest that the iron-fortified recombinant yeast strain is bioavailable.

• Journal of fish pathology
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• v.26 no.3
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• pp.295-301
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• 2013

Ferritin is an evolutionarily conserved protein that plays an important role in iron storage and detoxification. In this study, the gene encoding a ferritin H subunit homologue (RbFH) was cloned from rock bream (Oplegnathus fasciatus) and analyzed at the expression. The full-length ferritin H cDNA was 1162 bp long and contained an open reading frame (ORF) of 531 bp that encoded 177 amino acid residues with a predicted molecular mass of 20.8 kDa. The 5' UTR was 297 bp in length, and the 3' UTR 298 bp, and preceded by a 5'-untranslated region that contains a putative Iron Regulatory Element (IRE). The deduced amino acid sequence of RbFH shares extensive sequence identities with the H ferritins of a number of fish species and contains the ferroxidase center that is preserved in ferritin H subunits. Examination of tissue specific expression indicated that RbFH expression was most abundant in PBLs, RBC, liver and muscle.

• BMB Reports
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• v.34 no.4
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• pp.365-370
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• 2001

We constructed a comparative expression system in order to produce recombinant human ferritin homo- and heteropolymers in Escherichia coli. Human ferritin H-(hfH) and L-chain (hfL) genes were expressed without amino acid changes under the control of a tac promoter. Ferritin heteropolymers of varying subunit composition were also produced by combining two different expression systems, a bicistronic expression system and a coplasmid expression system. As a result, recombinant H-chain ferritin and ferritin heteropolymers were catalytically active in forming iron core in vivo. In particular, the ferritin heteropolymer that is composed of 7% H-subunit and 93% L-subunit was capable of forming an iron core of the protein, while the L-chain ferritin homopolymer was inactive in vivo. This result indicates that the two H-subunits (i.e., 7% H-subunit content) are important to keep ferritin active in the cells. In addition, human ferritins were identified as the major iron binding proteins in the transformed cells. Also, the amount of iron bound to the recombinant ferritins was proportional to the H-subunit content in ferritin heteropolymers in vivo.

• KSBB Journal
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• v.31 no.2
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• pp.105-112
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• 2016

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 $30^{\circ}C$, 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 $OD_{600}$ (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.

• Journal of Life Science
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• v.26 no.6
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• pp.698-704
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• 2016

The intracellular transport of organelles and protein complexes is mediated by kinesin superfamily proteins (KIFs). The first kinesin, kinesin 1, was identified as a molecular motor protein that moves various organelles and protein complexes along the microtubule rails in cells. Kinesin 1 is a tetramer of two heavy chains (KHCs, also called KIF5s) and two kinesin light chains (KLCs). KIF5s interact with many different proteins through their tail region, but their binding proteins have not yet been fully identified. To identify the interaction proteins for KIF5A, we performed yeast two-hybrid screening and found a specific interaction with ferritin heavy chain (Frt-h), which has a role in iron storage and detoxification. Frt-h bound to the amino acid residues between 800 and 940 of KIF5A and to other KIF5s in the yeast two-hybrid assay. The coiled-coil domain of Frt-h is essential for interaction with KIF5A. In addition, ferritin light chain (Frt-l) interacted with KIF5s in the yeast two-hybrid assay. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KHC specifically co-immunoprecipitated Frt-h and Frt-l from mouse brain extracts. These results suggest the kinesin 1 motor protein may transport the ferritin complex in cells.

• Nutritional Sciences
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• v.7 no.4
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• pp.218-222
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• 2004

Iron deficiency anemia (IDA) is the world's most common nutritional problem. It is characterized by a low hemoglobin (Hb) level and low iron status. A study was conducted to investigate the incidence of iron deficiency anemia in day scholar girls belonging to different socioeconomic strata at Punjab University, Lahore. Iron status of the subjects was estimated by measuring Hb, hematocrit (Hct), red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCR), serum iron, serum ferritin, total protein and albumin. Results indicated that females belonging to low socioeconomic strata had lower values for Hb, Hct, RBC count, total protein and albumin. Serum iron, serum ferritin, MCV and MCH values fell within the normal range for all of the socioeconomic groups. However, serum iron and ferritin varied with socioeconomic status and higher-income groups had significantly higher serum iron and ferritin. It was concluded that anemia may develop due to poor intake and absorption of iron and that those in the low-income bracket are the most affected group.

• Bulletin of the Korean Chemical Society
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• v.25 no.2
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• pp.237-242
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• 2004

Recombinant human ferritin homopolymers (rHF and rLF) were successfully produced in the Saccharomyces cerevisiae Y2805, which was transformed with human ferritin H or L-chain genes, respectively. In order to characterize the molecular properties of the recombinant ferritins in relation to mineralization, the proteins were isolated and apoferritins were prepared. The apoferritins were reconstituted with 2000 Fe atoms per protein molecule under various experimental conditions (the concentration of the protein, the buffer concentration of the MOPS buffer, the total volume of the reaction and the reconstitution method). The structure and composition of the iron cores formed in the ferritins were examined using transmission electron microscopy. The recombinant ferritins behaved in a similar manner to other mammalian ferritins in accumulating iron in the core. Proteins of rHF and rLF showed varying reconstitution yields of 37-72% depending on the reaction conditions. In general, the rHF showed higher reconstitution yield than the rLF at the protein concentrations and the reaction volumes we examined. Iron cores with a similar mean particle size were obtained in the rHF, rLF and horse spleen ferritin reconstituted at a protein concentration of 1.0 mg/mL. Electron diffraction of all the three ferritins showed 2-3 diffuse lines, with d-spacings corresponding to those of the mineral ferrihydrite with a limited crystallinity.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.299-307
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• 2008

Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by $Ni^{2+}$ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.