• Journal of Microbiology
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• v.35 no.2
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• pp.92-96
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• 1997

A new site-specific recombinase sin, as a component of a putatie transposon has been cloned and its base sequence has been determined. The proposed sin shows a hish degree of homology with pI9789-sin and pSK1-sin. There is a large (16 bp) inverted repeat downstream of proposed sin and the postulate dhelix-turn-helix motif is located at the extreme C-terminus of the poposed Sin. The transposase gene (tnpA) and .betha.-lactamase gene (blaZ) are located upstream of sin and arsenate reductase gene (arsC) and arsenic efflux pump protein gene (ars B) are downstream. This genetic arrangement seems to be a part of a new putative transposon because there is no known transposon with a gene arrangement of tnpA-blaZ-sin-arsC.

• KSBB Journal
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• v.28 no.5
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• pp.310-314
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• 2013

Antifreeze peptide from Myoxocephalus octodecemspinosus was overexpressed and purified in Escherichia coli. Green fluorescence protein-AFP chimera was constructed by integrating gfp and afp genes. Produced GFP-AFP chimera protein was purified using polyhistidine tag which was inserted at C-terminus. By addition of GFP-AFP chimera protein, freezing point of elution buffer was decreased from $-13^{\circ}C$ to $-20^{\circ}C$. This result suggested that GFP-AFP chimera can be considered as a potential candidate of novel inhibitor for gas hydrates.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.1-6
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• 1997

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

• Journal of Plant Biology
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• v.37 no.3
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• pp.349-355
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• 1994

Viral RNA was extracted from a purified orchid strain of tobacco mosaic virus (TMV-O) from Cymbidium "Grace Kelly". Polyadenylated viral RNAs were primed with Not I-oligo (dT) primer-adapter. First-strand cDNAs were reversely transcribed by Moloney murine leukaemia virus reverse transcriptase (RNAse H-), and then second-strand cDNAs were synthesized by RNase H and DNA polymerase I. The resulting double-stranded cDNAs were ligated into pSPORT1 vector and transformed into competent E. coli strain JM109 cells. The size of cDNAs within the recombinant plasmids was ranging from 0.9 to 3.9 kb. Among the selected clones, pTMO-0205 and -0210 covered the 3' half and the 5' half of the viral genomic RNA, respectively, which were covering more than 99% of the viral genemo size based on sequencing analysis. Two cDNA fragments which were 3.1 kb BamHI and NotI fragement released from pTMO-0.205 and 3.3 kb SalI and BamHI fragment released from pTMO-0210 were ligated with T4 DNA ligase. The clone was almost entire length, lacking only 31 nucleotides from the 5' terminus based on the sequencing result. This method was shown to be efficiently applicable to other plant viral gnomic RNA for the construction of cDNA.n of cDNA.

• BMB Reports
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• v.49 no.6
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• pp.337-343
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• 2016

Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce β-amyloid (Aβ), a key pathogenic molecule in Alzheimer's disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.244-251
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• 2001

Over half of all biologically active peptide and peptide hormones are $\alpha$-amidated at their C-terminus, which is essential for their full biological activities. Amidation is accomplished through the sequential reaction of the two enzymes encoded by the single bifunctional, peptidyl-glycine $\alpha$-amidating monooxygenase (PAM or an $\alpha$-amidating enzyme). PAM catalyze the forma - tion of a peptide amide from peptide precursors that include a C-terminal glycine, and requires copper molecular oxygen and ascorbate. PAM is the only enzyme that produces peptide amides in vivo. However various strategies utilizing PAM, carboxypeptidase-Y enzymes, and chemical syn-thesis have been developed for producing peptide amides in vitro. The growing need and impor-tance of peptide amide drugs has highlighted the necessity for a efficient in vitro amidating sys-tem for industrial application for the production of peptide hormones, like calcitonin and oxytocin. This review presents the current situation regarding amidation with a special emphasis on the in-dustrial production or peptide hormones.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.114-119
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• 1990

A yeast chromosomal superkiller gene (SK13) was cloned and expressed in $ski3^{-}$ Saccharomyces cerevisiae strains. The gene was fused to the structural region of E. coli lacZ gene at its C-terminus in a yeast-E. coli shuttle vector, pSR605. The fused gene complemented $ski3^{-}$ strains with SK13 activity and the quantitative level of expression was measured as determined by assaying $\beta$-galactosidase activity. The SDS-polyacrylamide gel electrophoresis and the Western blot analysis of this fused protein showed the immuno-reacted bands with a protein of the estimated molecular size (ca.250Kd).

• International Journal of Industrial Entomology and Biomaterials
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• v.10 no.2
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• pp.125-129
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• 2005

Ribosome-associated membrane protein 4 (RAMP4) is a membrane protein that exposes its N-terminal hydrophilic portion on the cytoplasmic side and spans the membrane close to the C-terminal end. RAMP4 has previously been reported to belong to the set of proteins that remains associated with membrane-bound ribosomes, and controls the glycosylation of major histocompatbility complex class II-associated invariant chain. RAMP4 also may be relative to the stabilization of membrane proteins in response to stress, with other components of translocon, and molecular chaperons in ER. Application of 5'-RACE technique with specially designed primer, we cloned a 715 bp cDNA fragment which contains a 195 bp ORF, termed RAMP4. The deduced protein has 64 amino acid residues and contains a putative transmembrane-spanning domain at the COOH terminus.

• BMB Reports
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• v.41 no.1
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• pp.86-91
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• 2008

The full-length cDNA of CaAbsi1 encodes a presumptive protein of 134 amino acid residues that has homology to a putative zinc finger protein in its C-terminus. The deduced amino acid sequence has 50% homology to Oryza sativa NP001049-274, the function of which is unknown. Expression of CaAbsi1 was reduced in response to inoculation of non-host pathogens. On the other hand it was induced one hour after exposure to high concentrations of NaCl or mannitol, and six hours after transfer to low temperature. Induction also occurred in response to oxidative stress, methyl viologen, hydrogen peroxide and abscisic acid. Our results suggest that CaAbsi1 plays a role in multiple responses to wounding and abiotic stresses.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.6
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• pp.728-735
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• 1995

The peptidyl prolyl cis-trans isomerase(PPlase, EC 5.2.2.8) from Bacillus stearothermophilus SIC1 was extracted from the cells treated with by lysozyme. PPlase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration (FPLC). The purity of purified the enzyme after Superose 12 column chromatography was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPlase was estimated as 18,000 by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0 to 8.0. The enzyme was considerably stable after heat treatment at $60^{\circ}C$ for 30 minutes, and the enzyme was quite stable up to $65^{\circ}C$. The presence of the PPlase in the refolding solution accelerated the isomerization rate of the assay peptide.