• 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.

• 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.

• 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.

• 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.

• BMB Reports
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• 제40권1호
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• pp.82-87
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• 2007

Our heterologous expression system of the human ferritin H-chain gene (hfH) allowed us to characterize the cellular effects of ferritin in yeasts. The recombinant Saccharomyces cerevisiae (YGH2) evidenced impaired growth as compared to the control, which was correlated with ferritin expression and with the formation of core minerals. Growth was recovered via the administration of iron supplements. The modification of cellular iron metabolism, which involved the increased expression of high-affinity iron transport genes (FET3 and FTR1), was detected via Northern blot analysis. The findings may provide some evidence of cytosolic iron deficiency, as the genes were expressed transcriptionally under iron-deficient conditions. According to our results examining reactive oxygen species (ROS) generation via the fluorescence method, the ROS levels in YGH2 were decreased compared to the control. It suggests that the expression of active H-ferritins reduced the content of free iron in yeast. Therefore, present results may provide new insights into the regulatory network and pathways inherent to iron depletion conditions.

• Plant Resources
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• 제1권1호
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• pp.13-21
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• 1998

A cDNA Fragment encoding iron storage protrin generated by polymerase chain reaction(PCR) using highly conserved regions of ferritin related genes were used to sereen a red pepper cDNA library. cDNA clone was designated as Fp1. Fp1 clone contatines a 5' nontranslated region of 51dp containing stop conds. Down stream from 5' UTP. an open reading frame of 750bp was observed. followed by a 3' UTR of 272bp. The deduces amino acid sequence of red pepper protein(Fp1) showed 84%, 48% and 36% identity with soybean(SolC). human(HuL H) and horse spleen(HoS-L) ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves. reaching a maxmum at 12h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.

• KSBB Journal
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• 제19권6호
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• pp.441-445
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• 2004

본 연구에서는 사람 ferritin H- 및 L-chain 유전자가 재조합된 효모 S. cerevisiae에 있어서 철의 생체 흡수 반응을 수행하였다. 재조합 효모는 $2\%$ galactose가 첨가된 YEP 배지에서 3일간 batch culture한 후, 20 mM MOPS buffer (pH 6.5) 에서 반응 균체 농도, 철 화합물 종류, 철 농도, 및 반응 시간 등을 고려하여 반응을 진행하였다. 이 실험 결과, ferritin H-chain 유전자를 발현하는 균주 YGH2에 있어서 균체 농도 100 mg/ml에서 균체 농도 200 mg/ml보다 높은 철 농도를 보였다. 그리고, 철 흡수 반응에 있어서 Fe(II)의 산화 상태가Fe(III)보다 훨씬 유리하였다. 철 농도의 증가에 따라 철 흡수량도 증가하였으며, 14.3 mM Fe(II)과 반응시 YGH2의 세포내 철 농도는 $16.7{\pm}0.7\;{\mu}mol/g$ cell wet wt.로 분석되었다. 철 흡수는 반응 시작 후 약 120분 경에 거의 최대치에 이르렀다.

• 대한미생물학회지
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• 제35권2호
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• pp.149-157
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• 2000

Mycobacterium tuberculosis is capable of growing and survival within macrophage. The purpose of this study was to identify the genes regulated by infection of mycobacteria in human monocytic THP-1 cells. We used the differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) and nothern blot analysis to confirm the differentially expressed genes from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv, heat-killed Mycobacterium tuberculosis H37Rv and live Mycobacterium bovis BCG. Among many up or down-regulated clones, 27 clones were sequenced and compared with known genes on GenBank. Thirteen of over-expressed clones from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv were identical to human prothymosin alpha, eight were novel clones and six clones showed homology with Human ferritin H chain, Esherichia coli bgl, Mouse RNA-dependent EIF-2 alpha kinase, E. coli htrL, Hyaluronan receptor and T cell receptor. Our result suggests that Mycobacterium tuberculosis might regulate prothymosin alpha gene transcription in monocytic THP-1 cell.