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Biomedical subjects

Bong Hyun Chung

Publications and source records attributed to Bong Hyun Chung.

At least 19 recordsLinked to original sources
PubMed2007

Oriented immobilization of antibodies with GST-fused multiple Fc-specific B-domains on a gold surface.

We have constructed a novel platform for the oriented buildup of immunoglobulins on a gold surface for a surface plasmon resonance imaging microarray. To this end, genetically engineered glutathione S-transferase proteins bearing one, two, and three Fc-specific B-domains in protein G from Streptococci (GST-GB1, -GB2, and -GB3, respectively) were produced. In order to tether these GST-GBx proteins specifically, a novel glutathione-derivatized ligand (LA-GSH) was also synthesized from a biaminated tri(ethylene glycol) backbone. Each end of the backbone was further functionalized with a maleimide group for a glutathione modification and a lipoic acid for surface immobilization. The glutathione ligand demonstrated a negligible nonspecific protein adsorption toward other spectator proteins while showing a strong specific association toward GST-GBx proteins. This Fc-specific surface exhibited at least a 2-fold enhancement in the immunoglobulin density (from human and mouse) with its antigen capture capability totally conserved compared to a covalently tethered GBx proteins. A single antibody tethered on the GST-GB3 is estimated to capture two antigens (enhanced green fluorescent protein), and this antigen capture ratio seems to be the most efficient value ever observed.

Animals↗
PubMed2007

A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system.

We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.

Escherichia coli↗
PubMed2006

Protein nanopatterns and biosensors using gold binding polypeptide as a fusion partner.

An efficient strategy for immobilizing proteins on a gold surface was developed by employing the gold binding polypeptide (GBP) as a fusion partner. Using the enhanced green fluorescent protein (EGFP), severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVme), and core streptavidin (cSA) of Streptomyces avidinii as model proteins, specific immobilization of the GBP-fusion proteins onto the gold nanoparticles and generation of protein nanopatterns on the bare gold surface were demonstrated. The GBP-fused SCVme bound to gold nanoparticles successfully interacted with its antibody and showed changes in absorbance and color, allowing efficient diagnosis of SARS-CoV. The fusion proteins could be successfully immobilized on the gold surface by nanopatterning and microcontact printing as examined by atomic force microscopy and surface plasmon resonance analysis. The poly(dimethylsiloxane) microfluidic channels were created on the gold surface and were used for antigen-antibody and DNA-DNA interaction studies. Specific immobilization of GBP-EGFP fusion protein and its interaction with the antibody in the microchannels could be demonstrated. By immobilizing the DNA probe through the use of GBP-fused cSA, specific hybridization of the target DNA prepared from Salmonella could also be achieved. The GBP-fusion method allows immobilization of proteins onto the gold surface without surface modification and in bioactive forms suitable for studying protein-protein, DNA-DNA, and other biomolecular interaction studies. Furthermore, these studies can be carried out in a microfluidic system, which allows high-throughput analysis of biomolecular interactions.

Biological Assay↗
PubMed2006

On-chip Escherichia coli culture, purification, and detection of expressed proteins.

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST-GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle.

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.

Coated Materials, Biocompatible↗
PubMed2006

Observation of self-assembled fluorescent beads by scanning near-field optical microscopy and atomic force microscopy.

Optical response and topography of fluorescent latex beads both on flat self-assembled monolayer and on a micron-patterned surface with poly(dimethylsiloxane) are studied. Scanning near-field optical microscopy and atomic force microscopy were utilized together for detecting fluorescence and imaging topography of the patterned latex beads, respectively. As a result, the micro-patterned latex beads where a specific chemical binding occurred show a strong signal, whereas no signals are observed in the case of nonspecific binding. With fluorescein isothiocyanate (FITC), it is convenient to measure fluorescence signal from the patterned beads allowing us to monitor the small balls of fluorescent latex.

Journal Article↗
PubMed2006

Surface plasmon resonance imaging analysis of protein-protein interactions using on-chip-expressed capture protein.

A surface plasmon resonance (SPR) imaging system, combined with a microwell gold chip for on-chip cell cultivation, was used to monitor protein-protein interactions. In particular, we developed an on-chip microscale cell cultivation system that integrates cell culture and on-chip analysis of protein-protein interactions on a single microwell chip in a time- and labor-saving manner. To assess the performance of this system in the analysis of protein-protein interactions, we conducted a series of protein-protein interaction analyses by measuring the binding of the yeast GAL4 dimerization domain (GAL4DD) to the GAL11 protein (GAL11P). Our system was found to enable the simple and rapid analysis of protein-protein interactions, requiring no special cell culturing equipment or recombinant protein expression prior to the immobilization of the purified proteins onto the chip. Our results demonstrate that the combination of an on-chip cell cultivation system and an SPR imaging system can be a useful tool to study protein-protein interactions without the need for time-consuming and labor-intensive protein preparation steps as well as fluorescent or other labeling of the interactants.

DNA-Binding Proteins↗
PubMed2006

Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53.

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.

Humans↗
PubMed2005

Monitoring of conformational change in maltose binding protein using split green fluorescent protein.

In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the reconstitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conformationally altered proteins. In particular, we used maltose binding protein (MBP) as a model protein, as MBP undergoes a characteristic hinge-twist movement upon substrate binding. The common feature of this approach is that GFP, as a reporter protein, splits into two non-fluorescent fragments, which are genetically fused to the N- and C-termini of MBP. Upon binding to maltose, the chromophores move closer together, resulting in the generation of fluorescence. This split GFP method also involves the reconstitution of GFP, which is determined via observations of the degree to which fluorescence intensity is restored. As a result, reconstituted GFP has been observed to generate fluorescence upon maltose binding in vitro, thereby allowing for the direct detection of changes in fluorescence intensity in response to maltose, in a concentration- and time-dependent fashion. Our findings showed that the fluorescence complementation assay can be used to monitor the conformational alterations of a target protein, and this ability may prove useful in a number of scientific and medical applications.

Carrier Proteins↗
PubMed2005

Detection of Bax protein conformational change using a surface plasmon resonance imaging-based antibody chip.

We describe an antibody chip technology that uses a surface plasmon resonance (SPR) imaging system to examine the conformational change of a protein. In this study, we used Bax protein, a pro-apoptotic member of the Bcl-2 family of proteins, as a model protein to investigate the conformational alteration triggered by a TNF-related apoptosis-inducing ligand (TRAIL), a potent inducer of apoptosis. To develop the antibody chip for detecting the Bax conformational change, we immobilized Bax monoclonal antibody 6A7, which recognizes only a conformationally changed Bax protein on a gold surface. The resultant immobilized Bax antibodies provided specific and accurate measurements of the active conformation-specific epitope in the apoptotic cancer cells treated with the TRAIL; these measurements corresponded to the data obtained by immunoprecipitation analysis using an active conformation-specific Bax antibody (6A7). The results of our study indicated that TRAIL-induced Bax structural change could be monitored quickly and simply using an SPR imaging system, thus demonstrating the potential for using such a system for the analysis of conformational properties of target proteins.

Antibodies↗
PubMed2005

Micropatterning proteins on polyhydroxyalkanoate substrates by using the substrate binding domain as a fusion partner.

A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (microCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein-protein interaction studies could be carried out using this system.

Adsorption↗
PubMed2005

Micropatterning of biomolecules on glass surfaces modified with various functional groups using photoactivatable biotin.

Biomolecule patterning by photolithographic methods has considerable advantages because a large number of different biomolecules can be assembled on a spatial area by a combinatorial method and complex biomolecule patterning can be created in situ in closed environments such as microfluidic channels. Here, a photobiotin was used as the photoactivatable reagent to create patterned arrays of biomolecules. The variability of photobiotin deposition on glass substrates modified with a variety of materials having carboxyl, lysine, aldehyde, amine groups, and BSA (bovine serum albumin) was characterized by subsequent derivatization with Cy3-labeled streptavidin. The fluorescence images of the photobiotin patterned glass surfaces showed that the BSA/aldehyde-coated glass could be considered as the most appropriate substrate to immobilize photobiotin, in view of the homogeneous immobilization of biomolecules with high density in defined regions and the reduction of nonspecific binding to the surface. In streptavidin equilibrium adsorption assays, the maximum amount of streptavidin-Cy3 bound to the BSA/aldehyde-coated glass surface continued to rise with increasing streptavidin-Cy3 concentration until 12.0 microg/mL was reached and the surface then became saturated. Also, a line array of biotin-labeled single-strand probe DNAs was created on the BSA/aldehyde-coated glass by photolysis of photobiotin through a slit-type mask and biotin/streptavidin/biotin chemistry, extended to a quantitative measurement of the concentrations of target DNA. The results of target DNA analysis showed linearity over a wide range from 0.5 ng/mL to 5 microg/mL and were reproducible.

Aldehydes↗
PubMed2005

Enhanced sensitivity of surface plasmon resonance (SPR) immunoassays using a peroxidase-catalyzed precipitation reaction and its application to a protein microarray.

We describe a method to improve the sensitivity of surface plasmon resonance (SPR) immunoassays using a horseradish peroxidase (HRP)-catalyzed precipitation reaction. The precipitation reaction catalyzed by HRP bound to the SPR biosensor surface via a sandwich immunoassay induced a shift in the SPR angle. Human interferon (IFN)-gamma at concentrations ranging from 0.01 to 100 ng/ml was detectable by this method. We also show that this biocatalytic signal amplification method can be applied to SPR imaging (SPRI), in an immunoassay of multiple proteins on a protein microarray format.

Catalysis↗
PubMed2005

Surface plasmon resonance imaging-based protein arrays for high-throughput screening of protein-protein interaction inhibitors.

The E7 protein produced by high-risk human papillomavirus (HPV) induces a degradation of the retinoblastoma tumor suppressor RB through direct interaction, which suggests that an inhibitor for the interaction can be a potential anticancer drug. A surface plasmon resonance (SPR) imaging-based protein array chip was developed for the high-throughput screening of inhibitor molecules targeting RB-E7 interaction. The glutathione S-transferase-fused E7 protein (GST-E7) was first layered onto a glutathionylated gold chip surface that had been designed to specifically bind to GST-fused proteins. Subsequently, a microarrayer was used to spot the hexa-histidine-tagged RB proteins (His(6)-RB) onto the GST-E7-layered gold chip surface, and the resulting SPR image was analyzed. Upon increased His(6)-RB concentration in the spotting solution, the SPR signal intensity increased proportionally, indicating that His(6)-RB bound to GST-E7 in a concentration-dependent manner. The His(6)-RB/GST-E7 interaction was challenged by spotting the His(6)-RB solution in the presence of a RB binding peptide (PepC) derived from a motif on E7. The SPR imaging data showed that PepC inhibited the His(6)-RB/GST-E7 interaction in a concentration-dependent manner. Our results show that the SPR imaging-based protein array chip can be applied to screen small molecule inhibitors that target protein-protein interaction.

Papillomavirus E7 Proteins↗
PubMed2005

Surface plasmon resonance imaging-based protein array chip system for monitoring a hexahistidine-tagged protein during expression and purification.

A surface plasmon resonance imaging-based Ni(2+)-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.

Escherichia coli↗
PubMed2004

A fusion protein expression analysis using surface plasmon resonance imaging.

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.

Affinity Labels↗