• Journal of Pharmaceutical Investigation
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• v.30 no.4
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• pp.253-258
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• 2000

In order to design the oral vaccine delivery system, we prepared the alginate micro spheres containing GFP (green fluorescent protein) as a model drug by spray method. To optimize the preparation conditions of microspheres, we investigated the effects of various parameters including nozzle pressure, nozzle opening angle, and concentrations of sodium alginate and calcium chloride. The prepared microspheres were evaluated by measuring their sizes, loading efficiency, and morphology. The particle size of microspheres was affected by the concentration of sodium alginate and calcium chloride, nozzle pressure, and nozzle opening angle. As the concentration of sodium alginate increased, GFP loading efficiency and particles size of microsphere also increased. However, it was observed to be difficult to spray the sodium alginate solution with concentration greater than 1.5% (w/v), due to high viscosity. The pressure over $3\;kgf/cm^2$ didn't affect the size of particles. As a result, the spraying method enabled us to prepare microspheres for oral vaccine delivery system. In this study, microspheres prepared with 1% (w/v) sodium alginate had greater loading efficiency and better spherical shape.

• Development and Reproduction
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• v.23 no.1
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• pp.55-61
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• 2019

To develop a promoter capable of driving transgene expression in non-model fish, we identified and characterized the muscle-specific alpha-actin gene in olive flounder, Paralichthys olivaceus (PoACTC1). The regulatory region of PoACTC1 includes putative regulatory elements such as a TATA box, two MyoD binding sites, three CArG boxes, and a CCAAT box. Microinjection experiments demonstrated that the regulatory region of PoACTC1, covering from -2,126 bp to +751 bp, just prior to the start codon, drove the expression of red fluorescent protein in developing zebrafish embryos and hatching olive flounder. These results suggest that the regulatory region of PoACTC1 may be useful in developing a promoter for biotechnological applications such as transgene expression in olive flounder.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.14-21
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• 2011

Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.

• KSBB Journal
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• v.30 no.6
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• pp.296-301
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• 2015

Eggshell-based biocomposites have become attractive due to their exquisite nanostructure and biological properties, which are mainly composed of highly organized calcium carbonate crystals controlled by organic macromolecules such as proteins and polysaccharides. Here, we designed the recombinant fusion protein of a putative eggshell matrix protein named as GG1234 and a fluorescent reporter protein of DsRed. The protein was successfully over-expressed in E. coli and purified by Ni-NTA affinity chromatography. In vitro calcium carbonate crystallization was conducted in the presence of the fusion protein, and morphological change was investigated. The protein inhibited the calcite growth in vitro, and spherical calcium carbonate micro-particles with the diameter of about $20-30{\mu}m$ were obtained. We expect that this study would be helpful for better understanding of eggshell-based biomineralization.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.15-23
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• 2000

The subcellular localization of the SopB protein, which is encoded by the Escherichia coli F plasmid and is involved in the partition of the single-copy plasmid, was directly visualized through the expression of the protein fused to the jellyfish green fluorescent protein (GFP). The fusion protein was found to localize to positions close but not at the poles of exponentially growing cells. Examination of derivatives of the fusion protein lacking various regions of SopB suggests that the signal for the cellular localization of SopB resides in a region close to its N terminus. Overexpression of SopB led to silencing of genes linked to, but well-separated from, a cluster of SopB-binding sites termed sopC. In this SopB-mediated repression of sopC-linked genes, all but the N-terminal 82 amino acids of SopB can be replaced by the DNA-binding domain of a sequence-specific DNA -binding protein, provided that the sopC locus is also replaced by the recognition sequence of the DNA-binding domain. These results suggest a mechanism of gene silencing: patches of closely packed DNA-binding protein is localized to specific cellular sites; such a patch can capture a DNA carrying the recognition site of the DNA -binding domain and sequestrate genes adjacent to the recognition site through nonspecific binding of DNA.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.306-311
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• 2005

In this study, the amino-terminal half truncated lump and the whole lump genes from Photobacterium phosphoreum coding for the lumazine protein were cloned by polymerase chain reaction and expressed in Escherichia coli. To identifiy of the binding site of the ligand or substrate, the amino acid identities from the sequences of the lumazine protein, yellow fluorescent protein, and riboflavin synthase from different organisms were also compared and analyzed.

• Proceedings of the Korean Aquaculture Society Conference
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• 2006.05a
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• pp.476-478
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• 2006

To analyze subcellular localization of betanodavirus protein B2, a plasmid expressing Betanodavirus protein B2 fused to enhanced green fluorescent protein (EGFP-Nl) was constructed. The transient expression of full-length B2 fused to EGFP in GF cells confirmed the equal distribution of protein B2 between cytoplasm and nucleus. However, transfection of N-terminal half of the B2 revealed that this truncated form predominantly localized to the cytoplasm. By using several deletion mutants and point mutants, we determined the regions and/or motif responsible for the subcellular localization of betanodavirus.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.630-633
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• 2003

Transfer of an electron from one site to another in a molecular or between molecules and/or electrodes is one of the most fundamental and ubiquitous processes in chemistry, biology and physics. In this study fusion proteins composed by green fluorescent protein(GFP) and cytochrome b562 were used in fabricating molecular array as an electron sensitizer and electron acceptor, Protein formation onto the substrate was performed by the self-assembly technique. The fusion protein film were analyzed using scanning probe microscope(SPM), Surface Plasmon Resornance(SPR) and hybrid STM/I-V. The results suggest that the proposed molecular photodiode can be used as a basic unit of the memory device.

• BMB Reports
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• v.43 no.12
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• pp.795-800
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• 2010

Huntingtin-interacting protein 1-related (HIP1r) is known to function in clathrin-mediated endocytosis and regulation of the actin cytoskeleton, which occurs continuously in non-dividing cells. This study reports a new function for HIP1r in mitosis. Green fluorescent protein-fused HIP1r localizes to the mitotic spindles. Depletion of HIP1r by RNA interference induces misalignment of chromosomes and prolonged mitosis, which is associated with decreased proliferation of HIP1r-deficeint cells. Chromosome misalignment leads to missegregation and ultimately production of multinucleated cells. Depletion of HIP1r causes persistent activation of the spindle checkpoint in misaligned chromosomes. These findings suggest that HIP1r plays an important role in regulating the attachment of spindle microtubules to chromosomes during mitosis, an event that is required for accurate congression and segregation of chromosomes. This finding may provide new insights that improve the understanding of various human diseases involving HIP1r as well as its fusion genes.