Biomedical subjects

Woo-Jae Chung

Publications and source records attributed to Woo-Jae Chung.

8 recordsLinked to original sources

PubMed2006

Selection of peptides for lipopolysaccharide binding on to epoxy beads and selective detection of Gram-negative bacteria.

Lipopolysaccharide (LPS)-binding peptides were enriched by using epoxy beads as a novel support to immobilize LPS for a phage displayed peptide library screening. The sequence of Phe-Ala-Pro-Trp (FAPW) was the most significant consensus motif of 10 selected clones, and Pro-Phe (PF) was the key dipeptide for binding at the apex of the loop to form a characteristic structure of CXXPFXXXC. Moreover, AWLPWAK, one of the highly conserved heptamer peptides, could detect specifically Gram-negative bacteria via a whole cell binding test at 10(6) cells ml(-1).

Bacterial Typing Techniques↗

PubMed2005

Combinatorial solid phase peptide synthesis and bioassays.

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.

Biological Assay↗

PubMed2005

Screening of LPS-specific peptides from a phage display library using epoxy beads.

The selection of identical or highly homologous peptides from phage display combinatorial peptide libraries has been unsuccessful in biopanning experiments using microtiter plates. In the present study, by biopanning on LPS-conjugated epoxy beads, we repeatedly enriched clones encoding AWLPWAK and NLQEFLF. These peptides were found to interact with the polysaccharide moiety of LPS, which is highly variable among gram negative bacterial species. In addition, phages encoding these peptides preferentially bound to the LPS of Salmonella family. AWLPWAK-conjugated beads absorbed Salmonella enteritidis from solution and showed a preference for S. enteritidis over Escherichia coli. In summary, this study shows for the first time that a peptide screened from phage displays of combinatorial peptide libraries can be synthesized on beads and be used practically to concentrate bacterial cells from solution.

Bacteriophages↗

PubMed2005

Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip.

A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated.

Biotin↗

PubMed2004

Core-shell-type resins for solid-phase peptide synthesis: comparison with gel-type resins in solid-phase photolytic cleavage reaction.

[reaction: see text] Novel core-shell-type resins with a rigid core and amino-functionalized flexible shell were prepared with 2,4,6-trichloro-1,3,5-triazine (CNC) and Jeffamine ED-600 starting from 1% cross-linked aminomethyl (AM) polystyrene resins. All of the amino groups were located outside the resin beads, and the loading capacity was 0.2-0.4 mmol/g. The amount of CNC treated was a determining factor in the properties of the final resins. The core-shell-type resins showed superior performances in terms of the initial loading of amino acid and the photocleavage reaction compared to the gel-type resins.

Catalysis↗

PubMed2004

Surface-grafted polystyrene beads with comb-like poly(ethylene glycol) chains: preparation and biological application.

We prepared surface-grafted polystyrene (PS) beads with comb-like poly(ethylene glycol) (PEG) chains. To accomplish this, conventional gel-type PS beads (35-75 microm) were treated with ozone gas to introduce hydroperoxide groups onto the surface. Using these hydroperoxide groups, poly(methyl methacrylate) (PMMA, Mn= 22,000-25,000) was grafted onto the surface of the PS beads. The ester groups of the grafted PMMA were reduced to hydroxyl groups with lithium aluminum hydride (LAH). After adding ethylene oxide (EO) to the hydroxyl groups, we obtained the PS-sg-PEG beads, which had a rugged surface and a diameter of 80-150 microm. We could obtain several kinds of the PS-sg-PEG beads by controlling the chain lengths of the grafted PMMA and the molecular weights of the PEG chains. The grafted PEG layer was about 30-50 microm thick, which was verified from the cross-sectioned views of the fluorescamine-labeled beads. These fluorescence images proved that the beads possessed a pellicular structure. Furthermore, we found that the surface-grafted PEG chains had the characteristic property of reducing non-specific protein adsorption on the beads.

Fluorescence↗

PubMed2004

Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker.

A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads. A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer. This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min. The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively. In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip. RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification. As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase. The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase.

Chromatography, Affinity↗

PubMed2003

Protein patterning by maskless photolithography on hydrophilic polymer-grafted surface.

With the help of a microfabrication process and surface modification technology, a method of fabricating protein patterned chips was developed which can be utilized as a powerful tool for performing bioassays in a high-throughput manner. A digital micromirror array (MMA) system was used as a virtual photomask, so that a maskless photolithography process was able to be used to build patterned biomolecules on a chip by selective illumination onto the chip surface. We utilized the nitroveratryloxycarbonyl (NVOC) group as a photolabile protecting group for protein patterning. The NVOC-protected surface was selectively irradiated by a UV illuminator using an MMA. After removing the NVOC group, biotin was coupled to the NVOC-cleaved site, onto which a buffered streptavidin solution was eluted. At this point, we could obtain a streptavidin-patterned surface and observe the effect of the polymer-grafted surface in reducing nonspecific binding.

Adsorption↗