• BMB Reports
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• v.36 no.6
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• pp.572-579
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• 2003

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of $\alpha$-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.

• Journal of Microbiology
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• v.44 no.1
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

• The Plant Pathology Journal
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• v.16 no.2
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• pp.110-117
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• 2000

Potato virus X (PVX-KO) showing mild mosaic and stunting symptoms on potato (Solanum tuberosum) in Kangwon area has been isolated and characterized. EM observation of the purified virus particles showed flexuous rod shape of about 520 nm in length. The coat protein (CP) of the virus had a molecular weight of 31 kDa in SDS-PAGE analysis, and the viral RNA was approximately 6.4 kb in size in denatured agarose gel electro-phoresis. In gel-immunodiffusion tests, it reacted strongly with an antiserum to common PVX from BIOREABAAG (USA). A rabbit antiserum was produced using purified virus and used for routine PVX detection by ELISA. Cultivated potatoes in Kangwon and other areas were frequently infected with PVX-KO. Both Datura stramonium and Nicotiana tabaccum cultivars developed necrotic local lesions 5 days after inoculation, and systemic mosaic symptoms with vein clearing 2 weeks after inoculation. All the features agree with the description of other PVX strains. To confirm and determine PVX strains, reverse transcription-polymerase chain reaction experiment was conducted using specific primers for viral CP. Amplified DNA fragments were cloned and sequenced. Results showed nucleotide sequence homologies of about 88 to 99% to other PVX strains. Based on CP amino acid sequence deduced from nucleotide sequences and host range studies PVX-KO is considered a member of the type X subgroup of PVX.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.226-230
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• 1999

To characterize the Korean isolate of infeciious hematopoietic necrosis virus (IHNV-PRT), a partial DNA fragment G gene of the MNV-PRT was amplified by RT-PCR. cloned inlo pGEM-T easy vector and analyzed for nucleotlde sequences. The size of the PCR pmduct was about 442 bp. The nucleotlde sequence homologies ofthe G gene of IHNV-PRT were 95%, 94%, 94% 94%, 93%, 53%. respectively. with those of foreign isolates of IHNV, IHNV-RB-76. IHNV-LR-73, MNV-K, IHNV-WRAC, Im-SRCV, IHNV-Col-85. However, it showed 81% homology with that of other fish rhabdovirus, hisame rhabdovirus (HRV). Frou~ the rcsults of deduced amino acid sequence homology analysis. G protein of IHNV-PRT showed 96% hornologies with those of foreign isolates of IHNV but 89% homology with that of HRV These results indicaled that, even though G gene of IHIW-PRT showed low homology with that of HRY it was highly conserved among different strains of THNV.

• Restorative Dentistry and Endodontics
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• v.22 no.2
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• pp.693-701
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• 1997

In 1992, Prevotella intermedia was shown to be comprised of another spoecies now known as Prevotella nigrescens. Strain ATCC 33563 is now designated the type strain of P. nigrescens while strain ATCC 25611 is remains the type strain of P. intermedia. The purpose of this study was to find the differences in protein profiles of P. intermedia and P. nigrescens, using sodium dodecyl sulfate polyacrylamide gel electrophoresis, which can be used for differentiation of those two species. A partial amino acid sequence of the 18.6 kDa protein band, which was specific in P. nigrescens, was also determined. The cellular proteins were extracted from the cell pellets of pure cultures of P. intermedia. and P. nigrescens by either sonication or being shaken continuously for 20 min at $21^{\circ}C$ with 1 % SDS or being boiled for 3 min with 1 % SDS. SDS-PAGE was performed according to the method of laemmli using either 12% (w/v) gels or 18% (w/v) gels. Results were as follows ; 1. The similar electrophoretic protein profiles were shown by 3 cellular protein extraction methods for each strain. (Fig. 1 and 2) 2. the 18.6 kDa band which was specific only in P. nigrescens could be used for the differentiation of P. intermedia. and P. nigrescens. (Fig. 1 and 2, Table 1) 3. A total of 4 different tryptic fragments from the 18.6 kDa protein were sequenced. the resulting amino acid sequences were fragment 1.GNPVNIGGEW, 2.FNVVR, 3.NYLT-VAPY, and 4.GGDNVTTYQVLPEIGYN. By comparison to the sequences of known proteins in the Swiss-Prot database and PIR database. 90 % matching between fragment 1 and serine hydroxymethyl transferase(P24060) in the Swiss-Prot, and 90% matching between fragment 1 and glycine hydroxymethyl transferase(S15203) in the PIR were shown, but the identity and function of the 18.6 kDa protein remains unknown.

• Journal of Mushroom
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• v.10 no.3
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• pp.120-128
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• 2012

Recently sawdust cultivation of Shiitake mushroom (Lentinula edodes) is increasing. It is important to make mycelia to be brown on the substrate surface. This browned surface in sawdust cultivation plays an important role like as artificial bark of the oak log, which protects the other pests and suppresses water evaporation in the substrate. In order to isolate genes which related to brown color formation, differential display method was used. Two cDNA fragments obtained by DD-PCR were 1.2 and 1.6kb and these were expressed in white colored mycelia from L. edodes, but not brown colored mycelia. Partial sequencing of these cDNA fragments showed that the 1.6kb cDNA had 100% identity with the microsatellites gene from Dugenia polichroa. However, the other 1.2kb cDNA fragment had poly T tail on 3' region of partial open reading frame on 5' region. The new primer designed based on the sequence of 1.2kb cDNA was constructed. RT-PCR analysis using the newly designed 0.12kb cDNA specific primer showed that the gene was only expressed in white color mycelia, but not in brown color mycelia. Sequence analysis of 5' region of this 1.2kb cDNA revealed that this gene contained partial open reading frame consisted of 110 amino acid. Homology search using DNASIS database showed that this gene had high sequence homology of 66.7% in DNA level and 69.2 % in amino acid level with dTDP-glucose 4,6-dehydratases gene from Arabidopsis thaliata. The dTDP-glucose 4,6-dehydratases gene was known to be function to have tolerance with oxidation stress. These results strongly suggest that this gene isolated from white mycelia of L. edodes might have a function of repressor against mycelia browning. Therefore I designated this gene as BCR (Brown Color Repressor) gene.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.13-18
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• 1990

A DNA sequence encoding the adr subtype preS2 region of hepatitis B virus envelope protein was fused to 5' end of lacZ gene yielding a plasmid pTSZ, in order to produce a preS2-$\beta$-galactosidase fusion protein. Serial deletions from 3' and 5' end of preS2 were constructed in plasmids, which were expressed and their antigenicities were examined with the monoclonal antibody H8. Deletions from amino and carboxy terminal to certain points did not affect the antigenicity, but the longer deletions destroyed the antigenicity. End points of deleted preS2 sequence were determined by DNA sequencing. As a result, each end of preS2 epitope was located in the region of amino acid residue 130-132 and 140-142, respectively. Residue 143 may be supplementary for antigenic epitope since the deletion from carboxy terminal to residue 143 revealed partial defect of antigenicity. In the interval of antigenic epitope the amino acid differences between adr and adw2 subtype occurred ar residue 130, 132, and 141. This result indicated that one or more of the three residues are responsible for the binding specificity of monoclonal antibody H8 to adr subtype preS2 fusion protein.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.102-102
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• 2001

Taurine (2-ethaneaminosulfonic acid) is one of the major intracellular ${\beta}$ -amino acids in mammals and is required for a number of biological processes including membrane stabilization, osmoregulation, antioxidation, detoxification, modulation of calcium flux and neurornodulation. The taurine transporter (TAUT) which contains 12 hydrophobic membrane-spanning domains has been cloned from dog kidney, rat brain, mouse brain, human thyroid, placenta and retina. In this study, The TAUT cDNA from the human intestinal epithelial cell, HT-29 was cloned and sequenced. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify partial cDNA encoding human intestinal TAUT. The coding region of the PCR product was 732 bp long. The primers were designed to encode highly conserved amino acid sequences near the transmembrane domains III (IPYFIFLF) and Ⅵ (KYKYNSYR) both in human and mouse. The TAUT cDNA amplified was ligated into the pGEX 4T-1 expression vector. The resulting sequence of human intestinal TAUT cDNA (Accession number of NCBI Genebank is AF346763) was identical to the sequences of the TAUTs previously determined in the human placenta and retina except 3 base pairs from that of the reported human thyroid. TAUT specific antibodies were generated to use them as biological tools in the studies of the biological role of TAUT. Peptides of 149-162 amino acid residue (14 amino acids) of the TAUT were synthesized. The synthetic peptide used in this study was LFQSFQKELPWAHC. This region was chosen not only to avoid putative glycosylation sites but also to exclude regions of known homology with GABA transporters in the extracellular hydrophilic domains. The synthetic peptide, TAUT-1 was conjugated with carrier protein, kehole lympet hemocyanin (KLH) to use as an antigen. When used for immunization on a rabbit to produce polyclonal antiserum, the conjugates elicited high -titered specific anti-TAUT-1 antibodies, which reacted well with the ovalbumin (OVA) conjugated peptides in ELISA. The KLH-conjugated peptide was also used as immunizing antigen in BALB/c mice to produce TAUT specific monoclonal antibodies. From the culture supernatant of the hybridoma, the specificity of anti-TAUT-1 monoclonal antibodies was confirmed by ELISA. Further applications of more tools in TAUT expression analysis will be performed such as western blotting and flow cytometry.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.3
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• pp.95-101
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• 2016

Apolipoprotein B-100 (Apo-B100) is a major component of low density lipoprotein (LDL). Apo B-100 protein has 4,536 amino acid sequence and these amino acids are classified into peptide groups A to G with subsequent 20 amino acids (P1-P302). The peptide groups were act as immunoglobulin (Ig) antigens which oxidized via malondialdehyde (MDA). The mimetic peptide P1 (EEEMLENVSLVCPKDAT RFK) out of D-group peptides carrying the highest value of IgG antigens were selected for structural studies that may provide antigen specificity. Circular Dichroism (CD) spectra were measured for peptide secondary structure in the range of 190-250 nm. Experimental results show that P1 exhibit partial of ${\beta}-sheet$ and random coil structure. Homonuclear (COSY, TOCSY, NOESY) 2D-NMR experiments were carried out for NMR signal assignments and structure determination for P1. On the basis of these completely assigned NMR spectra and distance data, distance geometry (DG) and Molecular dynamics (MD) were carried out to determine the structures of P1. The proposed structure was selected by comparisons between experimental NOE spectra and back calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P1 obtained upon superposition of all atoms was in the range $0.33{\AA}$. The solution state P1 has mixed structure of ${\beta}-sheet$ (Glu[1] to Cys[12]) and random coil (Pro[13] to Lys[20]). These NMR results are well consistent with secondary structure from experimental results of circular dichroism. Structural studies based on NMR may contribute to the studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.

• Korean Journal of Soil Science and Fertilizer
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• v.35 no.1
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• pp.59-67
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• 2002

A phosphate solubilizing bacterium. strain 60-2G, possessing a strong ability to solubilize insoluble phosphate was isolated from the rhizosphere of grass. On the basis of GC-FAME profile, carbon utilization pattern, and the DNA sequence of a conserved partial 16S rRNA gene, the 60-2G was identified as Enterobacter intermedium. The analysis by HPLC revealed that the strain 60-2G produced mainly gluconic and 2-ketogluconic acids with small amounts of lactic acid in broth culture medium containing hydroxyapatite. During the incubation period of the strain 60-2G in broth culture, pH of the medium decreased upto 3.8 while the soluble phosphate concentration increased. The reversed correlation between pH and soluble phosphate concentration indicated that the solubility of P was due to the produced organic acids. The sequence homology of the deduced amino acids suggested that E. intermedium 60-2G synthesized PQQ which is essential for the oxidation of glucose by glucose dehydrogenase.