• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.993-1001
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• 2007

The technique of serological analysis of antigens by recombinant cDNA expression library (SEREX) uses autologous patient sera as a screening probe to isolate tumor-associated antigens for various tumor types. Isolation of tumor-associated antigens that are specifically reactive with patient sera, but not with normal sera, is important to avoid false-positive and autoimmunogenic antigens for the cancer immunotherapy. Here, we describe a selection methodology to isolate patient sera-specific antigens from a yeast surface-expressed cDNA library constructed from 15 patient lung tissues with non-small cell lung cancer (NSCLC). Several rounds of positive selection using patient sera alone as a screening probe isolated clones exhibiting comparable reactivity with both patient and normal sera. However, the combination of negative selection with allogeneic normal sera to remove antigens reactive with normal sera and subsequent positive selection with patient sera efficiently enriched patient sera-specific antigens. Using the selection methodology described here, we isolated 3 known and 5 unknown proteins, which have not been isolated previously, but and potentially associated with NSCLC.

• Proceedings of the Microbiological Society of Korea Conference
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• 2004.05a
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• pp.42-44
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• 2004

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.278-284
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• 2007

A novel screening strategy for salt-resistant antimicrobial peptides from a M13 peptide library was developed. Fusion of MSI-344, a magainin derivative and indolicidin to pIII coat proteins did not significantly affect viability of the recombinant phages, which indicated that the pIII could neutralize toxicity of the antimicrobial peptides and therefore it is possible to construct antimicrobial peptide library in Escherichia coli. On the basis of the conserved sequence of ${\alpha}$-helical antimicrobial peptides, a semi-combinatorial peptide library was constructed in which the peptides were displayed by pIII. To remove hemolytic activity from the library, the phages bound to red blood cells were removed, and the subtracted phage library was screened for binding to target bacteria Pseudomonas aeruginosa and Staphylococcus aureus under high salt concentrations. The screened peptides showed relatively low antimicrobial activity against the target bacteria. However, antimicrobial activities of the screened peptides P06 and S18 were not affected by the cation concentrations of 150 mM $Na^+$, 2 mM $Mg^{2+}$ and 2 mM $Ca^{2+}$ without significant hemolytic activity. This screening strategy that is based on binding capacity to target cells provides new potential to develop salt-tolerant antimicrobial peptides.

• Animal cells and systems
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• v.5 no.1
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• pp.65-69
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• 2001

Obtaining mutant animals is important for studying the function of a particular gene. A chemical mutagenesis was first carried out to generate mutations in C. elegans. In this study, we used ultraviolet-activated 4,5',8-trimethylpsoralen to induce small deletion mutations. A library of mutagenized worms was prepared for recovery of candidate animals and stored at $15^{\circ}C$ during screening instead of being made into a frozen stock library. In order to isolate deletion mutations in target genes, a polymerase chain reaction (PCR)-based screening method was used. As a result, two independent mutants with deletions of approximately 1.0 kb and 1.3 kb were isolated. This modified and improved reverse genetic approach was proven to be effective and practical for isolating mutant animals to study gene function at the organismal level.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.779-784
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• 2013

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an anti-leptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1696-1701
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• 2010

The ${\alpha}$-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis ${\alpha}$-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.

• Biomedical Science Letters
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• v.15 no.2
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• pp.105-112
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• 2009

The screening of the anti-cancer activity of the chemical library provided 2-thio-4-aminopyrimidine as the initial hit. The confirmation of structure and biological effect of hit was performed by synthesis and biological evaluation. The optimization of hit was performed by derivatization of substituents while keeping the core structure. The evaluation of growth inhibitory activity was carried out using SRB assay against 6 human cancer cell lines and human fibroblast. The growth inhibitory activity of compounds showed substituent dependency and more than 5 compounds showed complete growth inhibition of cancer cell lines at 10 ${\mu}M$ concentration. Chemical library screening followed by synthetic modification provided possibility that 2-thio-4-aminopyrimidine can be used as a new scaffold for the development of anti-cancer agent.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.134-138
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• 2005

The design of ion channel targeted library is a valuable methodology that can aid in the selection and prioritization of potential ion channel-likeness for ion-channel-targeted bio-screening from large commercial available chemical pool. The differences of property profiling between the 93 ion-channel active compounds from MDDR and CMC database and the ACDSC compounds were classified by suitable descriptors calculated with preADME software. Through the PCA, clustering, and similarity analysis, the compounds capable of ion channel activity were defined in ACDSC compounds pool. The designed library showed a tendency to follow the property profile of ion-channel active compounds and can be implemented with great time and economical efficiencies of ligand-based drug design or virtual high throughput screening from an enormous small molecule space.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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• pp.521-529
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• 2016

Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.

• BMB Reports
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• v.38 no.5
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• pp.517-525
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• 2005

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.