• Title/Summary/Keyword: C-terminus

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Papaya Ringspot Virus Coat Protein Gene for Antigen Presentation in Escherichia coli

  • Chatchen, Supawat;Juricek, Mila;Rueda, Paloma;Kertbundit, Sunee
    • BMB Reports
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    • v.39 no.1
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    • pp.16-21
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    • 2006
  • The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.

Expression Properties and Skin Permeability of Human Basic Fibroblast Growth Factor with or without PTD Fused to N- or C-terminus in Escherichia coli (대장균 발현시스템에서 단백질 전달 도메인 PTD가 인간 섬유아세포 성장인자(FGF2)의 N- 또는 C-말단에 결합 되었을 때 미치는 재조합 단백질 복합체의 발현 특성과 피부 투과능력)

  • Park, In-Sun;Choe, Chung-Hyeon;Kwon, Bo-Ra;Choi, Young-Ji;Kwon, Tae-Ho;Yu, Kang-Yeol;Lee, Juhyung;Choo, Young-Moo
    • Journal of Life Science
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    • v.28 no.3
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    • pp.275-283
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    • 2018
  • Human fibroblast growth factor (FGF) has the potential to be a commercially important therapeutic or cosmeceutical agent due to its ability to generate tissue and heal wounds. Granting permeability into skin tissues increases the therapeutic effects of FGF. Thus, several researchers have attempted the fusion of FGF conjugates with protein transduction domains (PTDs) to investigate the transduction ability and therapeutic effects of FGF. Less is known, however, about whether the location of PTD fused to the N- or C-terminus of FGF proteins has a significant impact on the folding and stability in Escherichia coli, and eventually, on transduction. Here, we report cloning of human basic fibroblast growth factor (FGF2) as a control and FGF2 with PTD fused to the N- or C-terminal ends of FGF proteins by an overlap extension PCR. We performed expression, verified expression properties of recombinant FGF2 without or with PTD fused to the N-terminus and the C-terminus, and investigated transduction ability into tissue by treating the dorsal skin of mice subjects. As a result, FGF2 and FGF2-PTD (fused to C-terminus) fusion protein were expressed as soluble forms suitable for straight-forward purification, unlike insoluble PTD-FGF2 (fused to N-terminus), but only FGF2-PTD fusion protein could transduce into the dorsal skin tissue of the mice subjects. Our results suggest that FGF2 with PTD fused to the C-terminus is more efficient than other options in terms of expression, purification, and delivery into skin tissue, as it does not require labor-intensive, costly, and time-consuming methods.

Membrane interaction of the coiled-coil motif of HIV gp41 and its implication in the membrane fusion process

  • Jin, Bong-Suk;Yu, Yeon-Gyu
    • Proceedings of the Korean Biophysical Society Conference
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    • 2003.06a
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    • pp.58-58
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    • 2003
  • The envelope glycoprotein of HIV, gp41, mediates the membrane fusion with human cells. The extracellular domain of gp41 has two helical regions. The N-terminus helical region (N-helix) forms trimeric coiled coil, interacts with the C-terminus helical region (C-helix) of gp41 to form a stable helical bundle structure. In this study, we have shown that the N-helix of gp41 has membrane interacting and disrupting abilities. It was localized into the interface of the lipidic phase and head group of the membrane. In contrast, the N-helix region with membrane fusion defective mutations could not bind to membrane. In addition, the N-helix bound on the membrane was released from the membrane by the C-helix, and the complex of the N- and C-helix did not interact with membrane. These results suggested that the membrane binding ability of the N-helix is necessary for the fusion activity of gp41, and such property is possibly controlled by the C-helm.

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Identification of Proteins Interacting with C- Terminal Region of Human Ankyrin-G

  • Lee, Yeong-Mi;Lee, Min-A;Park, Jae-Kyoung;Kim, Myong-Shin;Jeon, Eun-Bee;Park, Su-Il;Kim, Chong-Rak
    • Biomedical Science Letters
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    • v.9 no.3
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    • pp.159-165
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    • 2003
  • Ankyrins are a ubiquitously expressed family of intracellular adaptor proteins involved in targeting diverse proteins to specialized membrane domains in both the plasma membrane and the endoplasmic reticulum. Recently, the studies with C-terminus of ankyrins have identified that ankyrin-B is capable of interacting with Hsp40 and sAnkl is capable of interacting with obscurin and titin, but the function of C-terminal domain of ankyrin-G remains unknown. To identify proteins interacting C-terminus of ankyrin-G, we used the C-terminus of ankyrin-G as a bait for a yeast two-hybrid screen of brain cDNA library. Approximately 1.33$\times$l0$^6$ transformants were screened, of which 13 positive clones were obtained as determined by activation of HIS3, ADE2 and MELl reporter genes. Sequence analyses of these 13 plasmids revealed that cDNA inserts of 13 colonies showed highly homologous to 11 genes, including 5 known (i.e., Na$^+$/K$^+$ ATPase $\beta$1, SERBPl, UTF2, cytochrome C oxidase and collagen IV $\alpha$2) and 6 unknown genes. The evaluation of the proteins that emerge from these experiments provides a rational approach to investigate the those proteins significant in interaction with ankyrin-G.

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Molecular interaction of $G{\alpha}16$ subunit G protein with chemoattractant receptors

  • Ha, Ji-Hee;Lee, Chang-Ho
    • Proceedings of the PSK Conference
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    • 2002.10a
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    • pp.275.1-275.1
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    • 2002
  • It has been proposed that G protein interacts with receptor via multiple interaction sites. With regard to this, C-terminus of the G${\alpha}$ subunit is clearly not the only structural determinant on the G proteins that is critical for receptor coupling selectivity, but the extreme N-terminus of Ga subunit and other structural elements were proposed to be responsible for dictating the interaction with receptors. (omitted)

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The PcG protein hPc2 interacts with the N-terminus of histone demethylase JARID1B and acts as a transcriptional co-repressor

  • Zhou, Wu;Chen, Haixiang;Zhang, Lihuang
    • BMB Reports
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    • v.42 no.3
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    • pp.154-159
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    • 2009
  • JARID1B (jumonji AT rich interactive domain 1B) is a large nuclear protein that is highly expressed in breast cancers and is proposed to function as a repressor of gene expression. In this paper, a phage display screen using the N-terminus of JARID1B as bait identified one of the JARID1B interacting proteins, namely PcG protein (Polycomb group) hPc2. We demonstrated that the C-terminal region, including the COOH box, was required for the interaction with the N-terminus of JARID1B. In a reporter assay system, co-expression of JARID1B with hPc2 significantly enhanced the transcriptional repression. These results support a role for hPc2 acting as a transcriptional co-repressor.

The Antitumor Effect of C-terminus of Hsp70-Interacting Protein via Degradation of c-Met in Small Cell Lung Cancer

  • Cho, Sung Ho;Kim, Jong In;Kim, Hyun Su;Park, Sung Dal;Jang, Kang Won
    • Journal of Chest Surgery
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    • v.50 no.3
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    • pp.153-162
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    • 2017
  • Background: The mesenchymal-epithelial transition factor (MET) receptor can be overexpressed in solid tumors, including small cell lung cancer (SCLC). However, the molecular mechanism regulating MET stability and turnover in SCLC remains undefined. One potential mechanism of MET regulation involves the C-terminus of Hsp70-interacting protein (CHIP), which targets heat shock protein 90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, we investigated the functional effects of CHIP expression on MET regulation and the control of SCLC cell apoptosis and invasion. Methods: To evaluate the expression of CHIP and c-Met, which is a protein that in humans is encoded by the MET gene (the MET proto-oncogene), we examined the expression pattern of c-Met and CHIP in SCLC cell lines by western blotting. To investigate whether CHIP overexpression reduced cell proliferation and invasive activity in SCLC cell lines, we transfected cells with CHIP and performed a cell viability assay and cellular apoptosis assays. Results: We found an inverse relationship between the expression of CHIP and MET in SCLC cell lines (n=5). CHIP destabilized the endogenous MET receptor in SCLC cell lines, indicating an essential role for CHIP in the regulation of MET degradation. In addition, CHIP inhibited MET-dependent pathways, and invasion, cell growth, and apoptosis were reduced by CHIP overexpression in SCLC cell lines. Conclusion: C HIP is capable of regulating SCLC cell apoptosis and invasion by inhibiting MET-mediated cytoskeletal and cell survival pathways in NCI-H69 cells. CHIP suppresses MET-dependent signaling, and regulates MET-mediated SCLC motility.

Nebulin C-terminus Interacts with NCBP51, a New Isoform of RING Finger Protein 125 (RNF125)

  • Kim, Ji-Hee;Kim, Hyun-Suk;Park, Eun-Ran;Choi, Jae-Kyoung;Lee, Yeong-Mi;Choi, Jun-Hyuk;Shin, Jung-Woog;Kim, Chong-Rak
    • Biomedical Science Letters
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    • v.13 no.1
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    • pp.1-10
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    • 2007
  • Nebulin, a giant modular protein from muscle, is thought to act as molecular ruler in sarcomere assembly. In skeletal muscle, the C-terminal ${\sim}50 kDa$ region of nebulin extends into the Z-line lattice. The most recent studies implicated highlighting its extensive isoform diversity and exciting reports revealed its expression in cardiac and non-muscle tissues containing brain. Also these novel findings are indicating that nebulin is actually a multifunctional filament system, perhaps playing roles in signal transduction, contractile regulation, and myofibril force generation, as well as other not yet defined functions. However the binding protein of nebulin and function in brain is still unknown. A novel binding partner of nebulin C-terminal region was identified by screening a human brain cDNA library using yeast two-hybrid system. Nebulin C-terminus binding protein 51 (NCBP51) was contained a RING-finger domain and identified a new isoform of RING finger protein 125 (RNF125). The interaction was confirmed using the GST pull-down assay. NCBP51 belongs to a family of the RING finger proteins and its function remains to be identified in brain. The role of nebulin and NCBP51 will be studied by loss-of-function using siRNA technique in brain.

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Periplasmic Expression of a Recombinant Antibody (MabB9) in Escherichia coli

  • Chang, Hae-Choon;Kwak, Ju-Won
    • Journal of Microbiology and Biotechnology
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    • v.7 no.5
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    • pp.299-304
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    • 1997
  • Expression in the periplasm of Escherichia coli of cloned heavy and light chain cDNAs for Fab fragment of a murine monoclonal antibody MabB9 (${\gamma}2b$, K), specific for human plasma apolipoprotein B-100 of LDL, was studied. For the purpose, a vector for two-cistronic expression of the heavy chain cDNA, at the 5' terminus, and light chain cDNA, at the 3' terminus, was constructed using the signal sequences, pelB (for heavy chain) and ompA (for light chain) in a pET vector system. The constructed vector was transformed into E. coli BL21(DE3). The expressed heavy chain (25 kDa) and light chain (23 kDa) of the antibody molecule were detected in total cell extracts as well as in the periplasmic extracts of E. coli.

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Green Tea Catechin Causes an Weight Loss in Transgenic Mice Over-expressing Carboxyl Terminus of Amyloid Precusor Protein

  • Lim Hwa-J.;Kim Yong-K.;Sheen Yhun-Y.
    • Biomolecules & Therapeutics
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    • v.14 no.2
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    • pp.96-101
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    • 2006
  • Amyloid$\beta(A{\beta})$ has been reported have an effect on the induction of oxidative stress that involves the functional and structural abnormalities in Alzheimer's disease. As a role of a radical scavenger, a green tea treatment was found have some inhibitory effect on the neurodegenerative process. The aim of this study was to determine if green tea catechin (GTC) reduces in transgenic model. To test this, transgenic mice carrying neuronspecific enolase (NSE) controlled C-terminus (105) of APP (APP-C105) were created and treated them with a low ana high dose of GTC for 6 months. Herein, we conclude that transgenic mice expressing NSE/APP-C105 were successfully created and the GTC-treated group exhibited significant reduction in body weight. Thus, GTC might be a good prevention of obesity or good treatment for AD patient.