• KSBB Journal
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• v.26 no.2
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• pp.119-125
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• 2011

In the synthesis of nanoparticles, much attention has been paid to regulating the particle size. There has been a possible evident that using the central cavity (core) of the protein ferritin has a greatly significant influence on it because the core can generate the nanometer-sized mineral particles of variable metal ions. In this report, recombinant human L-ferritins produced from Saccharomyces cerevisiae were purified and their molecular properties were characterized. The cDNA for human ferritin L chain was also expressed in another host such as Escherichia coli, and the properties of recombinant L-ferritins were compared. From isoelectric focusing experiment, the L-ferritin from the recombinant yeast showed no indication of N-glycosylation. Some post-translational modifications other than N-glycosylation were speculated in the L-ferritins from yeast. A difference was made in the L-ferritins in their iron uptake rates and the initial rate of the L-ferritin from yeast was slightly increased. The reconstitution yield and size distribution of the core minerals were analyzed in the L-ferritins by transmission electron microscopy. The L-ferritin from yeast with higher reconstitution yield (54.5%) showed slightly larger sizes (mean 6.92 nm) with narrower size distribution than the L-ferritin from E. coli. It is, in conclusion, speculated that L-ferritin from yeast is relatively superior to the other, in view of the size of nanoparticle and its relative homogeneity.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.926-932
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• 2008

Human heavy chain (H-) and light chain (L-) ferritins were amplified from a human cDNA library. Each ferritin gene was inserted downstream of the T7 promoter of bacterial expression vectors, and two types of coexpression vectors were constructed. The expression levels of recombinant ferritins ranged about 26-36% of whole-cell protein. H-ferritin exhibited a lower expression ratio compared with L-ferritin, by a coexpression system. However, the coexpression of HL-ferritins was significantly increased above the expression ratio of H-ferritin by cultivation without IPTG induction overnight. Purified recombinant H-, L-, HL-, and LH-ferritins were shown to be homo- and heteropolymeric high molecular complexes and it was indicated that their assembled subunits would be able to work functionally in the cell. Thus, these results indicate an improvement in the expression strategy of H-ferritin for heteropolymeric production and studies of ferritin assembly in Escherichia coli.

• 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.

• Bulletin of the Korean Chemical Society
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• v.29 no.10
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• pp.1969-1972
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• 2008

In this study, horse spleen apoferritins were induced to form biominerals using up to 3000 Fe atoms per protein molecule. The morphology and crystallinity of the nanometer-sized biominerals formed in the ferritins were then analyzed using field emission-energy filtering-transmission electron microscopy (FE-TEM). The ferritins were found to have reconstitution yields of 60-70% in the experiments. The mean core size of the ferritins varied somewhat with protein concentrations, indicating that crystal growth in ferritins could be controlled via protein concentrations. The core mineral size increased with the amount of Fe used. Lattice fringes of the core, associated with good crystallinity, were found in all samples. The lattice fringe images of a single domain ferrihydrite mineral appeared frequently in the (011) planes (d-spacing of 0.246 nm) under [100] zone axis in all samples of this study. In addition, the lattice image occasionally revealed fringes corresponding to the (100) planes (d = 0.254 nm) from the [001] zone axis, indicating the characteristic pattern of hexagonal crystal lattice. Diffraction patterns in the minerals identified as ferrihydrite were fitted well into the space group of $P3_{1c}$.

• BMB Reports
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• v.29 no.6
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• pp.540-544
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• 1996

Ferritins from germinated pumpkin seeds were isolated by ammonium sulfate precipitation (0.55 saturation), ion-exchange chromatography on DEAE-cellulose, and gel filtration chromatographies on Sephacryl S-300 and Sephadex G-100. Pumpkin ferritin contains less iron than soybean ferritin. Pumpkin ferritin cross-reacted with anti-soybean ferritin antiserum made in rabbit, and showed two distinct antibody reactive bands, both of equal intensity. The pumpkin ferritins corresponding to the two bands were separable by centrifugation in a sucrose gradient (20~50%). The molecular weights of the native pumpkin ferritins based on the estimation of sucrose gradient centrifugation, gel filtration on Sephacryl S-300 and non-denaturing polyacrylamide gel electrophoresis appeared to be: 530~580 KD (the large molecular weight pumpkin ferritin) and 330-360 KD (the small molecular weight pumpkin ferritin) The large molecular weight pumpkin ferritin contains less iron. Both pumpkin ferritins cross-reacted with anti-soybean ferritin antibody with a spur formation suggesting partial antigenic recognition.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1695-1699
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• 2007

Ferritin is an iron storage protein found in most living organisms as a natural assembled macromolecule. For studying the functional ability of the ferritin assembly, human H- and L-ferritins were expressed and purified from Pichia pastoris strain GS115. The recombinant H- and L-ferritins showed a globular form with transmission electron microscopy. The rate of iron uptake for H-ferritin was significantly faster than that for the L-ferritin in vitro. By gel permeation chromatography analysis, recombinant ferritins were confirmed as multimeric subunits with high molecular weight and it was indicated that assembled subunits were able to store iron in vivo.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.221-226
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• 2008

To produce human ferritins in yeast, human H-chain and L-chain ferritins were amplified from previously cloned vectors. Each amplified ferritin gene was inserted into the pYES2.1/V5-His-TOPO yeast expression vector under the control of the GAL1promoter. Western blot analysis of the recombinant yeast cells revealed that H-and L-chain subunits of human ferritin were expressed in Saccharomyces cerevisiae. Atomic absorption spectrometry (AAS) analysis demonstrated that the intracellular content of iron in the ferritin transformant was 1.6 to 1.8-fold higher than that of the control strain. Ferritin transformants could potentially supply iron-fortified nutrients for food and feed.

• 한국정보디스플레이학회:학술대회논문집
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• 2009.10a
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• pp.553-556
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• 2009

We investigated an application of supramolecular protein, and demonstrated the metal induced lateral crystallization utilizing ferritins with Ni nanoparticles, named the "bio-nano-crystallization". So far, this method has required long time, because of this method condition based on the conventional solid phase crystallization. In this study, we applied the pulsed rapid thermal annealing to bio-nanocrystallization. As a result, we succeeded in the crystallization for a short time. We found that the TFTs characteristics were improved with decrease metal impieties in poly-Si thin films by this method.

• 한국전자현미경학회:학술대회논문집
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• 1996.05a
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• pp.59-62
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• 1996

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1368-1376
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• 2008

In our previous study, the expression of active H-ferritins in Saccharomyces cerevisiae was found to reduce cell growth and reactive oxygen species (ROS) generation upon exposure to oxidative stress; such expression enhanced that of high-affinity iron transport genes (FET3 and FTR1). The results suggested that the recombinant cells expressing H-ferritins induced cytosolic iron depletion. The present study analyzes metabolic changes under these circumstances via proteomic methods. The YGH2 yeast strain expressing A-ferritin, the YGH2-KG (E62K and H65G) mutant strain, and the YGT control strain were used. Comparative proteomic analysis showed that the synthesis of 34 proteins was at least stimulated in YGH2, whereas the other 37 proteins were repressed. Among these, the 31 major protein spots were analyzed via nano-LC/MS/MS. The increased proteins included major heat-shock proteins and proteins related to endoplasmic reticulum-associated degradation (ERAD). On the other hand, the proteins involved with folate metabolism, purine and methionine biosynthesis, and translation were reduced. In addition, we analyzed the insoluble protein fractions and identified the fragments of Idh1p and Pgk1p, as well as several ribosomal assembly-related proteins. This suggests that intracellular iron depletion induces imperfect translation of proteins. Although the proteins identified above result from changes in iron metabolism (i.e., iron deficiency), definitive evidence for iron-related proteins remains insufficient. Nevertheless, this study is the first to present a molecular model for iron deficiency, and the results may provide valuable information on the regulatory network of iron metabolism.