• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.110-111
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetrating peptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells (Fig. 1). Fig. 1. Fluorescent microscopic images of SKBR3 breast cancer cells (A~C) and MCF10A breast cells (D~F) treated with Cy3-trastuzumab/fFcBP-Pf_Fn complexes. Trastuzumab and FcBP-Pf_Fn, which were labeled with Cy3 (Cy3-trastuzumab) and fluorescein (fFcBP-Pf_Fn), respectively, selectively targeted SKBR3 over MCF10A.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.84-84
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetratingpeptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells.

• Molecules and Cells
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• v.26 no.3
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• pp.270-277
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• 2008

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.8
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• pp.1089-1095
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• 2012

Neonatal Fc receptor (FcRn) gene encodes a receptor that binds the Fc region of monomeric immunoglobulin G (IgG) and is responsible for IgG transport and stabilization. In this report, the 8,900 bp porcine FcRn genomic DNA structure was identified and putative FcRn protein included 356 amino acids. Alignment and phylogenetic analysis of the porcine FcRn amino acid sequences with their homologies of other species showed high identity. Tissues expression of FcRn mRNA was detected by real time quantitative polymerase chain reaction (Q-PCR), the results revealed FcRn expressed widely in ten analyzed tissues. One single nucleotide polymorphism (SNP) (HQ026019:g.8526 C>T) in exon6 region of porcine FcRn gene was demonstrated by DNA sequencing analysis. A further analysis of SNP genotypes associated with serum Classical Swine Fever Virus antibody (anti-CSFV) concentration was performed in three pig populations including Large White, Landrace and Songliao Black pig (a Chinese indigenous breed). Our results of statistical analysis showed that the SNP had a highly significant association with the level of anti-CSFV antibody (At d 20; At d 35) in serum (p = 0.008; p = 0.0001). Investigation of expression and polymorphisms of the porcine FcRn gene will help us in further understanding the molecular basis of the antibody regulation pathway in the porcine immune response. All these results indicate that FcRn gene might be regarded as a molecular marker for genetic selection of anti-CSFV antibody level in pig disease resistance breeding programmes.

• IMMUNE NETWORK
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• v.24 no.1
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• pp.1.1-1.6
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• 2024

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.342-347
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• 2007

Bacteriophage ${\Phi}FC1$ integrase (MJ1) was previously shown to perform a site-specific recombination between a phage attachment site (attP) and a host attachment site (attB) in its host, Enterococcus faecalis, and also in a non-host bacterium, Escherichia coli. Here, we investigated biochemical features of MJ1 integrase. First, MJ1 integrase could perform in vitro recombination between attP and attB in the absence of additional factors. Second, MJ1 integrase interacted with att sites. Electrophoretic mobility shift assays and DNase I footprinting revealed that MJ1 integrase could efficiently bind to all the att sites and that MJ1 integrase recognized relatively short sequences (${\sim}50bp$) containing an overlapping region within attB and attP. These results demonstrate that MJ1 integrase indeed catalyzes an integrative recombination between attP and attB, the mechanism of which might be simple and unidirectional, as found in serine integrases.

• Microbiology and Biotechnology Letters
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• v.39 no.4
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• pp.337-344
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• 2011

Integrase MJ1 from the bacteriophage ${\Phi}FC1$ carries out recombination between two DNA sequences (the phage attachment site, attP and the bacterial attachment site, attB) in NIH3T3 mouse cells. In this study, the integration vector containing attP, attB and the integrase gene MJ, was constructed. The integration mediated by integrase MJ1 in Escherichia coli led to excision of LacZ. Therefore, the frequency of integration was measured by the counting of the white colony, which is detectable on X-Gal plates. The extrachromosomal integration in NIH3T3 mouse cells was monitored by the expression of the green fluorescent protein (GFP) as a reporter. To demonstrate integration mediated integrase MJ1 in NIH3T3 cells, vectors containing attP and attB were co-transfected into NIH3T3 cells. The integration was confirmed by fluorescent microscopy. The expression of GFP was induced in NIH3T3 cells expressing MJ1 without accessory factors. By contrast, the excision mediated by the MJ1 between attR and attL had no effect on the expression of GFP. These results suggest that integrase MJ1 may enable a variety of genomic modifications for research and therapeutic purposes in higher living cells.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.105-111
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• 1999

The effects of exogenous spleen cells on the progesterone and insulin like-growth factor-I (IGF-I) secretions in luteal cells were studied by using in vitro luteal cell culture system in the Hanwoo luteal cells. The corpora lutea(CL) were collected and pooled from the Korean native cattle(Hanwoo) ovaries from a local slaughter house. After enzymatic dissociation, combined large and small luteal cells(LLC and SLC)(1.0$\times$10$^{6}$ cells/$m\ell$) were incubated in D-MEM media containing antibiotics and 10% FCS. Spleen cells (1.0$\times$10$^{6}$ cells/$m\ell$) obtained from castrated adult male Hanwoo were added to luteal cells and co-cultured for 24 h in the absence or presence of luteinizing hormone (LH) (100 ng). Progesterone contents from luteal tissues were increased at CL-3 stage during each stage of estrous cycle. Progesterone secretion from luteal cell culture by the presence of LH (100 ng/$m\ell$) was positively stimulated compared with control. However, progesterone secretion was not changed by the addition of 5, 10 and 20% of spleen cells in the absence of LH. Co-culture of luteal cells with 10% of spleen cells in the presence of LH(l00ng/$m\ell$) significantly. enhanced after 24 h of culture. IGF-Isecretion from in vitro luteal cells co-culture by the addition of spleen cells (5%, 10% and 20%) was not significantly effected. Besides, in the presence of LH (100ng/$m\ell$), IGF-Isecretions from luteal cells by addition of spleen cells were higher than control media. However, LH alone significantly increased IGF-I secretion at 24 h of culture. These data provide the demonstrate that spleen cells can enhance LH action so as to stimulate progesterone secretion from Hanwoo luteal cells but have no effect to stimulate IGF-I secretion.