• KSBB Journal
• /
• 제17권4호
• /
• pp.370-375
• /
• 2002

새로운 유전자를 클로닝하고 그 발현양상을 결정하는 것은 유전자의 기능을 이해하는데 필수적이다. 인간유전자 12q13의 고해상 물리지도를 작성하면서 이 지역의 D12S359와 D12S1618 사이에 내재하는 것으로 mapping된 stSG 3435 EST의 유전자를 클로닝하고 그 발현양상을 조사하였다. NIBI library를 조사하여 stSG 3435를 포함하는 클론 325E4를 분리하여 순차적 결실 방법으로 클로닝하여 자동염기서열분석으로 염기서열을 결정하였다. 1,331 bp의 염기서열을 가진 이 유전자는 Blast search에 의하면 376 개의 아미노산으로 이루어진 단백질로써 인간의 MYGI과 동일하며 마우스의Gamml, melanocyte proliferation gene 1과 86%의 동질성을 보였다. MYGI은 인간염색체의 12에 내재하며 마우스의 Gamml은 syntenic 부위인 마우스 염색체 15에 내재하므로 마우스의 Gamml의 homolog으로 간주된다. Northern blot analysis 결과 MYG1은 인간의 모든 조직에서 발현되며 정소에서 가장 강한 발현을 보였다. 이 유전자의 세포내 발현을 green fluorescence protein과 융합시켜 발현 귀착지를 confocal 현미경으로 동정한 결과 MYG1 단백질은 핵과 리소좀을 제외한 소기관에서 발현되는 것을 관찰하였다.

• Bulletin of the Korean Chemical Society
• /
• 제25권2호
• /
• pp.237-242
• /
• 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.

• BMB Reports
• /
• 제35권5호
• /
• pp.476-481
• /
• 2002

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae $\alpha$-factor or Pem-CMG was employed. The results demonstrated that ${\alpha}Pem$-CMG, either with (${\alpha}2EACMG$) or without (${\alpha}CMG$) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either ${\alpha}2EACMG$ or ${\alpha}CMG$ was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both ${\alpha}2EACMG$ and ${\alpha}CMG$ was correctly processed. This produced the mature Pem-CMG peptide.

• BMB Reports
• /
• 제36권3호
• /
• pp.294-298
• /
• 2003

The pathological effect of the Bacillus thuringiensis Cry $\delta$-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

• Molecules and Cells
• /
• 제37권7호
• /
• pp.562-567
• /
• 2014

Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-${\beta}1$. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.

• BMB Reports
• /
• 제41권9호
• /
• pp.651-656
• /
• 2008

A mouse with cataract, Kec, was generated from N-ethyl-N-nitrosourea (ENU) mutagenesis. Cataract in the Kec mouse was observable at about 5 weeks after birth and this gradually progressed to become completely opaque by 12 weeks. Dissection microscopy revealed that vacuoles with a radial or irregular shape were located primarily in the cortex of the posterior and equatorial regions of the lens. At the late stage, the lens structure was distorted, but not ruptured. This cataract phenotype was inherited in an autosomal recessive manner. We performed a genetic linkage analysis using 133 mutant and 67 normal mice produced by mating Kec mutant (BALB/c) and F1 (C57BL/6 $\times$ Kec) mice. The Kec locus was mapped to the 3 cM region encompassed by D14Mit34 and D14Mit69. In addition we excluded coding sequences of 9 genes including Rcbtb2, P2ry5, Itm2b, Med4, Nudt15, Esd, Lcp1, Slc25a30, and 2810032E02Rik as the candidate gene that causes cataract in the Kec mouse.

• BMB Reports
• /
• 제35권2호
• /
• pp.206-212
• /
• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• Molecules and Cells
• /
• 제19권3호
• /
• pp.328-333
• /
• 2005

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as $70^{\circ}C$. Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.

• BMB Reports
• /
• 제28권2호
• /
• pp.143-148
• /
• 1995

In E. coli, chromosomal DNA associated with proteins is condensed into an organized structure known as nucleoid. Using a nitrocellulose filter binding assay to identify proteins forming nucleoid, a 21 kDa protein was purified from E. coli. The molecular weight of the purified protein was 21 kDa on SDS-polyactylamide gel electrophoresis and 24 kDa on gel permeation chromatography. A molecular weight of 21 kDa on SDS-polyacrylamide gel electrophoresis is unique among known proteins which are believed to be involved in the formation of nucleoid in E. coli. The 21 kDa protein nonspecifically binds to both double-stranded and single-stranded DNA. Sedimentation in a sucrose gradient revealed that the protein induced significant condensation of both supercoiled plasmid DNA and linear bacteriophage $\lambda$ DNA On the basis of quantitative Western-blot analysis, approximately 40,000 molecules of the protein were estimated to exist in an E. coli. The biochemical properties and cellular abundance of the 21 kDa protein suggest that this protein participates in the formation of nucleoid in E. coli.

• KSBB Journal
• /
• 제19권5호
• /
• pp.394-398
• /
• 2004

A derivatization method in mass spectrometry for small molecular analysis was developed to solve the problems of volatility of many analytes, difficult ionization of analytes, and undiscriminating isobaric analytes. This derivatization method, oximation of $\alpha$-ketoacid, in the transaminase reaction leads to change of mass difference between amine reactants and ketoacid products. In addition, regardless of the kinds of ketoacid, the linear relationship between the peak intensity and its concentration (from 1 mM to 10 mM) shows that quantitative analysis of the conversion of the reaction can be executed by the analysis of peak intensity of the corresponding oximated ketoacids. Furthermore, this method can be used for identifying transaminase as well as determining its substrate specificity