• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.68-73
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• 2004

Ferritin is an iron storage protein found in most living organisms. For expression and industrial use, human light chain ferritin (L-ferritin) was cloned from human liver cDNA library and expressed in Pichia pastoris strain GS115. The recombinant L-ferritin in Pichia pastoris was glycosylated. In a fed-batch culture, the cell mass reached about 57 g/l of dry cell weight, and the L-ferritin in the cell was increased to about 95 mg/l after 150 h. In an atomic absorption spectrometry analysis, the intracellular content of iron in the L-ferritin transformant was measured as $1,694{\pm}85\;\mu\textrm{g}g/g$, which is 5.4-fold more than that of the control strain. This L-ferritin transformant could serve as iron-fortified nutrients in animal feed stock.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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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.

• KSBB Journal
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• 제26권2호
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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.

• BMB Reports
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• 제34권4호
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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.

• 한국미생물·생명공학회지
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• 제36권3호
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• pp.221-226
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• 2008

효모에서 사람의 H-, L-ferritin을 생산하기 위해서, 기존에 복제된 vector를 사용하였으며,단백질을 발현하기 위해서 각각의 증폭된 ferritin 유전자를 GALI promoter에 의해 조절되는 pYES2.1/V5-His-TOPO 효모 발현 vector에 삽입하였다. Western blot 분석을 통해서 사람의 H-, L-ferritin subunits을 함유한 재조합 효모에서 사람의 ferritin이 발현된 것을 확인할 수 있었다. 또한 Atomic absorption spectrometry(AAS) 분석을 통해서 형질변환된 효모의 철 함유량이 대조군과 비교하여 $1.6{\sim}l.8$배 증가한 것을 확인하였다. 향후 ferritin이 함유된 형질변환 효모를 사용하여 잠재적으로 철이 강화된 영양성분을 기능성 식품과 사료에 이용할 수 있을 것이다.

• 동아시아식생활학회지
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• 제16권1호
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• pp.93-98
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• 2006

This study examined whether or not the iron that is accumulated in the recombinant microbes that produce ferritin is bioavailable to rats with iron deficiency. Rats induced with iron deficiency were treated with iron preparations of $Fe(NH_4)_2(SO_4)_2$, horse spleen ferritin, control yeast, and ferritin-producing recombinant yeast for 14 days. The bioavailability of iron was examined by measuring hemoglobin concentration, hematocrit value, and tissue iron stores. Differences between dietary groups were determined by one-way ANOVA, at the level of significance p<0.05. Based on hemoglobin concentration and hematocrit value, iron in $Fe(NH_4)_2(SO_4)_2$, horse spleen ferritin, and ferritin-producing yeast were bioavailable in rats and cured iron deficiency. The efficacy of ferritin and ferritin-producing yeast was confirmed in establishing tissue iron stores after the induction of iron deficiency. The iron sources of ferritin and the ferritin-producing yeast seemed to be as effective for the recovery from iron deficiency as the iron compounds of ferric citrate and ferrous ammonium sulfate. The results suggest that the iron stored in ferritin of the recombinant yeast is bioavailable, and that the recombinant yeast may contribute widely as a source of iron to resolve the global problem of iron deficiency.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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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.

• KSBB Journal
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• 제17권2호
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• pp.162-168
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• 2002

본 연구에서는 유도성 promoter인 GAL1과 상시성 promoter인 GPD와 ADH1 promoter 하류에 사람 ferritin H-chain 유전자(hfL) 및 사람 ferritin L-chain 유전자(hfL)를 연결하여 재조합 plasmid를 구축하고 이들을 S. cerevisiae 2805에 형질 전환시켜 외래 유전자를 성공적으로 발현시켰다. Ferritin 발현에 미치는 promoter의 영향을 비교한 바, 이 두 단백질 생간에 있어서 GAL1 promoter가 GPD나 ABH1 promoter 보다 더 효율적이었다. GAL1 promoter를 사용한 형질 전환체 (YG-H와 YG-L)에서 H-chain의 발현율은 전체 수용성 단백질 중 4.5%에 해당하였고, L-chain의 발현율은 9.8%에 이르렀다. 각 균주에서 발현된 H 및 L subunit은 비변성 젤은 사용한 전기 영동의 결과 대장균에서 생산된 재조합 단백질과 마찬가지로 자발적으로 holoprotein으로 조합되어졌다. 재조합 H-와 L-ferritin들은 단백질 내공에 철을 축적할 수 있음이 Prussian blue 염색으로 확인되었다. 그리고 효모 세포를 10 mM ferric citrate를 함유한 배지에서 배양했을 때, H-ferritin과 L-ferritin을 생산하는 재조합 효모 균주에 있어서의 펄의 농도는 각각 174.9 $\mu\textrm{g}$ Per gram(dry cell weight)과 148.8 $\mu\textrm{g}$ Per gram(dry cell weight)이었고 야생형 효모 균주에 있어서의 털의 농도는 49.4 $\mu\textrm{g}$ Per gram(dry cell weight)이었다. 이것은 사람 ferritin 유전자를 효모 균주에 발현시킴으로써 효모의 철 함량이 증진되었음을 유추하는 결과이다.

• KSBB Journal
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• 제19권5호
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• pp.352-357
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• 2004

Human ferritin H- and L-chain genes (hfH and hfL) were cloned into the yeast shuttle vector YEp352 containing the GAL1 (galactokinase) and GAL10 (epimerase) divergent promoters and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. SDS-PAGE displayed expression of the introduced hfH and hfL in both recombinant strains of Y1H10L and Y1L10H. The ferritin subunits, that represented ca. $22\%$ and $15\%$ of the soluble proteins in Y1H10L and Y1L10H, were spontaneously assembled into active ferritin heteropolymers. The H subunit content of the purified recombinant human ferritin heteropolymers was proven to reflect the relative expression yield of the subunits. When the cells of 2d culture were incubated with 14.3 mM Fe(2), the cellular iron concentration of Y1H10L and Y1L10H was 1.7 and 2.0 times, respectively, that of the control strain. It is assumed that increase in the iron uptake of the recombinant yeasts is closely related to ferritin expression and H subunit content.

• BMB Reports
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• 제29권5호
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• pp.411-416
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• 1996

In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.