• The Korean Journal of Physiology
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• v.22 no.2
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• pp.163-177
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• 1988

The $Ca^{2+}-ATPase$ of sarcoplasmic reticulum (SR) was solubilized and reconstituted into a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios in order to assess the effect of physical states of phospholipids on the incorporation and functions $Ca^{2+}-ATPase$. On the basis of the spectral data of Ca-arsenazo III, the $Ca^{2+}$ uptake of SR was increased linearly as the PC content increased in the reconstituted vesicles. The ATP hydrolysis activity also increased as PC content increased up to 25% and then decreased slightly as the PC content further increased. On the other hand the incorporation of $Ca^{2+}-ATPase$ into the reconstituted vesicls occured maximally at 25% PC and 75% PE mixture which is known to have a non-bilayer structure in reconstitution system. From the above results it is clear that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution and lamellar structure of the bilayer facilitates the $Ca^{2+}-ATPase$ function.

• KSBB Journal
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• v.16 no.2
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• pp.146-152
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• 2001

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we propose a solid-phase refolding method integrated with expanded bed adsorption chromatography. The model protein used was a fusion protein of recombinant human growth hormone and a glutathione S transferase fragment. It was demonstrated that the EBA-mediated refolding technique could simultaneously remove cellular debris and directly renature the fusion protein inclusion bodies in the cell homogenate with much higher yields and less agregation. To demonstrate the applicability of the method, we successfully tested the three representative types of starting materials, i. e., rhGH monomer, washed inclusion bodies, and the E. coli homogenate. This direct and simplified refolding process could also reduce the number of renaturation steps required and allow refolding at a higher concentration, at approximately 2 mg fusion protein per ml of resin. To the best of our knowledge, it is the first approach that has combined the solid-phase refolding method with expanded bed chromatography.

• Journal of Life Science
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• v.21 no.12
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• pp.1778-1783
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• 2011

Hematopoietic cytokines regulate production of blood cells by stimulating proliferation and differentiation of bone marrow cells. Among these hematopoietic cytokines, called hematopoitic growth factors, glranulocyte-colony stimulating Factor (G-CSF), which regulates growth of neutrophils, is one of important therapeutic factors because cancer patients suffer with neutropenia which is severe reduction of neutrophils after chemotherapy. Two groups of recombinant G-CSF have approved and used for therapeutic purposes and many researches are still on-going to produce recombinant G-CSF by different techniques. We engineered human G-CSF with Bombyx specific endoplasmic reticulum (ER) signal sequence, therefore, secretion of human G-CSF protein was improved in Bombyx mori-origined cell line, Bm5. The Bombyx ER signal sequence and human G-CSF matured protein region chimera was further remodeled at the N-terminus of matured G-CSF protein to understand roles of N-terminus on outer cellular secretion and/or production. Three different mutants were generated deleting three amino acids in non alpha-helical region in N-terminus in order to scan important amino acids for G-CSF secretion. One of 3 different N-terminal deletion mutants showed dramatically reduction of secreted amount of G-CSF indicating its important role on secretion. The data suggest that remodeling in non alpha-helical region of N-terminus is also important for recombinant G-CSF production.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.45-51
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• 2014

A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Korean Journal of Poultry Science
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• v.48 no.3
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• pp.111-122
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• 2021

Recently, interleukin 34 (IL-34) was identified as the second functional ligand for macrophage colony-stimulating factor receptor (M-CSFR). IL-34 functions similarly to M-CSF through its binding to the M-CSFR. There is still insufficient information on IL-34 in chickens, which has until now been reported only through predicted sequences and not through experimental research. Thus, to confirm its expression and to determine its potent biological activity, several chicken lines and cell lines were used. Cloning of recombinant chicken IL-34 and M-CSF genes was performed to investigate their modulatory effects on proinflammatory cytokine expression in vitro. The expression levels of IL-34, M-CSF, and M-CSFR genes were upregulated in broiler chickens with leg dysfunction (cause unknown). However, IL-34 was downregulated in most pathogen-stimulated tissues. M-CSFR expression was enhanced by recombinant IL-34 and M-CSF proteins in vitro. IFN-γ expression was enhanced by recombinant IL-34, but not by M-CSF. However, IL-12 expression was not regulated in any of the treated cells, and IL-1β was decreased in all tissues. These results indicate that IL-34 and M-CSF have roles in both the classical and alternative macrophage activation pathways. Collectively, our findings demonstrate the expression of IL-34 in chickens for pathogenic trials, both in vitro and in vivo. Our results suggest that the IL-34 protein plays a role in both pro- and anti-inflammatory functions in macrophages. Therefore, further research is needed to determine the cytokines or chemokines that can be induced by IL-34 and to further elucidate the functions of IL-34 in the inflammatory pathway.

• KSBB Journal
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• v.17 no.2
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• pp.162-168
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• 2002

Human ferritin H- and L-chain genes(hfH and hfL) were cloned into the yeast shuttle vector YEp352 with various promoters, and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. Three different promoters fused to hfH and hfL were used: galactokinase 1 (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase(GPD) promoter and alcohol dehydrogenase 1(ADH1 ) promoter. SDS-polyacrylamide gel electrophoresis and Western blotting analyses displayed expression of the introduced hfH and hfL. In the production of both ferritin H and L subunits GAL1 promoter was more effective than GPD promoter or ADH1 promoter. Ferritin H and L subunits produced in S. cerevisiae were spontaneously assembled into its holoproteins as proven on native polyacrylamide gels. Both recombinant H and L-chain ferritins were catalytically active in forming iron core. When the cells were cultured in the medium containing 10 mM ferric citrate, the cell-associated concentration of iron was 174.9 $\mu\textrm{g}$ Per gram(dry cell weight) for the recombinant yeast YG-L and 148.8 $\mu\textrm{g}$ Per gram(dry cell weight) for the recombinant yeast YG-L but was 49.4 $\mu\textrm{g}$ Per gram(dry cell weight) in the wild type, indicating that the iron contents of yeast is improved by heterologous expression of human ferritin H-chain or L-chain genes.

• Research in Plant Disease
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• v.18 no.3
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• pp.231-235
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• 2012

Indian citrus ring spot virus (ICRSV) is known to cause a serious disease to citrus, especially to Kinnow mandarin, the popular cultivated citrus species in India. In this study, we developed diagnostic systems based on enzyme-linked immunosorbent assay (ELISA). In order to generate antibodies against ICRSV coat protein, we overexpressed the coat protein in Escherichia coli using the pET15b expression vector containing an optimized ICRSV coat protein gene. The recombinant ICRSV coat protein was overexpressed as soluble form at $37^{\circ}C$ upon IPTG induction. The protein was purified to 95% in purity by Ni-NTA column chromatography. The purified protein was immunized to rabbit for the generation of polyclonal antibody (PAb). The PAb showed a specific immunoreaction to recombinant ICRSV coat protein in western blot analysis and ELISA. Diluted rabbit antisera (10,000 fold) could detect less than 10 ng and 5 ng of the target protein in western blot and ELISA analysis, respectively.

• Analytical Science and Technology
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• v.12 no.6
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• pp.565-570
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• 1999

The alcohol dehydrogenase (ADH) gene from Bacillus stearothermopilus was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pGEX-KG, and expressed it as a fusion protein with glutathione S-transferase (GST) in E. coli. The recombinant ADH was produced by induction with 1 mM isopropyl-${\beta}$-D-thiogalactopyranoside at $37^{\circ}C$ and purified by glutathione affinity chromatography. The recombinant ADH exhibited high substrate specificity for ethanol. The activity of the recombinant ADH proceeded optimally at pH 9.0 and $70^{\circ}C$. The recombinant ADH was highly stable against high temperature. This thermostable alcohol dehydrogenase can be used for the enzymatic determination of alcohol and for the industrial production of alcohol.

• Journal of Sericultural and Entomological Science
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• v.52 no.2
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• pp.102-109
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• 2014

We constructed the fibroin H-chain expression system to produce enhanced yellow fluorescent proteins (EYFP) in the silk of transgenic silkworm. Fluorescent silk could be made by fusing EYFP cDNA to the heavy chain gene and injecting it into a silkworm. The EYFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EYFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The yellow fluorescence proving that the fusion protein was present in the silk. Accordingly, we suggest that the EYFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.