• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.211-214
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• 2013

Bisphenol A (BPA) is a highly hazardous component to human since it is regarded as one of endocrine disruptors. For the analysis and/or removal of BPA, the searching for the specific ligand with a selective affinity to target BPA is required. In order to find the peptide moiety that specifically binds to BPA, the ultrasound-assisted biopanning was carried out with a phage-displayed peptide library expressing constrained heptamer. After six rounds of positive screening against BPA particles followed by the negative screening against the surface of eppendorf tube, the peptide sequence (CysLysSerLeuGluAsnSerTyrCys) with affinity to BPA was screened based on the order of frequency from the screened phage clones. To further verify the specificity of screened peptide sequence, the cross-binding affinity of the phage peptide toward BPA analogues such as Bisphenol S (BPS) and Bisphenol F (BPF) was also assessed, where the selected phage peptide showed a higher affinity to BPA over BPS and BPF.

• Journal of Life Science
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• v.7 no.3
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• pp.167-171
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• 1997

As an initial effort to elucidate RNA: protein binding in a way to regulate translation initiation and phosphorylation, a cDNA encoding a double-stranded RNA binding factor (RBFII)was isolated from Hela ZAPII cDNA library by affinity screening using [$\alpha$$^{-32}$P] UMP-labeled HIV Rev-responsive element(RRE) RNA. The nucleotide sequence of RBF (or TRBP) cDNA except the 5’end. At the 5’end, This common ORF was fused in-frame to N-terminal residues of Lac-Z through a unique 138 nt sequence encoding 46residues in the case of RBFII and a 63 nt sequence encoding 21 residuces in the case of RBFI. The context of ATG appearing first in the sequences suggests that both these cDNA inserts are incomplete at the 5’end.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.3
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• pp.1571-1576
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• 2016

Alteration of the acetylation status of chromatin and other non-histone proteins by HDAC inhibitors has evolved as an excellent epigenetic strategy in treatment of cancers. The present study was sought to identify compounds with positive pharmacological profiles targeting HDAC1. Analogues of Vorinostat synthesized by Cai et al, 2015 formed the test compounds for the present pharmacological evaluation. Hydroxamte analogue 6H showed superior pharmacological profile in comparison to all the compounds in the analogue dataset owing to its better electrostatic interactions and hydrogen bonding patterns. In order to identify compounds with even better high affinity and pharmacological profile than 6H and Vorinostat, virtual screening was performed. A total of 83 compounds similar to Vorinostat and 154 compounds akin to analogue 6H were retrieved. SCHEMBL15675695 (PubCid: 15739209) and AKOS019005527 (PubCid: 80442147) similar to Vorinostat and 6H, were the best docked compounds among the virtually screened compounds. However, in spite of having good affinity, none of the virtually screened compounds had better affinity than that of 6H. In addition SCHEMBL15675695 was predicted to be a carcinogen while AKOS019005527 is Ames toxic. From, our extensive analysis involving binding affinity analysis, ADMET properties predictions and pharmacophoric mappings, we report Vorinostat hydroxamate analogue 6H to be a potential candidate for HDAC inhibition in treatment of cancers through an epigenetic strategy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.3759-3765
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• 2015

Background: Approaches in disruption of MDM2-p53 interactions have now emerged as an important therapeutic strategy in resurrecting wild type p53 functional status. The present study highlights virtual screening strategies in identification of high affinity small molecule non-peptidic inhibitors. Nutlin3A and RG7112 belonging to compound class of Cis-imidazoline, MI219 of Spiro-oxindole class and Benzodiazepine derived TDP 665759 served as query small molecules for similarity search with a threshold of 95%. The query molecules and the similar molecules corresponding to each query were docked at the transactivation binding cleft of MDM2 protein. Aided by MolDock algorithm, high affinity compound against MDM2 was retrieved. Patch Dock supervised Protein-Protein interactions were established between MDM2 and ligand (query and similar) bound and free states of p53. Compounds with PubCid 68870345, 77819398, 71132874, and 11952782 respectively structurally similar to Nutlin3A, RG7112, Mi219 and TDP 665759 demonstrated higher affinity to MDM2 in comparison to their parent compounds. Evident from the protein-protein interaction studies, all the similar compounds except for 77819398 (similar to RG 7112) showed appreciable inhibitory potential. Of particular relevance, compound 68870345 akin to Nutlin 3A had highest inhibitory potential that respectively showed 1.3, 1.2, 1.16 and 1.26 folds higher inhibitory potential than Nutilin 3A, MI 219, RG 7112 and TDP 1665759. Compound 68870345 was further mapped for structure based pharamacophoric features. In the study, we report Cis-imidazoline derivative compound; Pubcid: 68870345 to have highest inhibitory potential in blocking MDM2-p53 interactions hitherto discovered.

• BMB Reports
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• v.29 no.3
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• pp.205-209
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• 1996

To investigate the role of heparin in keratinocyte growth factor (KGF) and acidic fibroblast growth factor (aFGF) high affinity binding to the KGF receptor (KGFR), a cell free system was established which utilized a secreted chimeric molecule between the KGFR extracellular domain and the immunoglobulin heavy chain Fc domain (KGFR-HFc). KGFR-HFc was purified from NIH 3T3 cells and demonstrated the binding of $[^3H]-heparin$ as well as heparin Sepharose. Scatchard analysis showed that the dissociation constant for heparin binding to KGFR-HFc was 140 nM. High affinity KGF and aFGF binding to KGFR-HFc remained unchanged after treatment with 0.6 M NaCl, which is the concentration sufficient to release any bound heparin to the KGFR-HFc. These results strongly suggest that although the KGFR interacts with heparin, the presence of heparin is not absolutely required for high affinity binding of either KGF or aFGF to the KGFR.

• Journal of Information Technology Applications and Management
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• v.27 no.4
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• pp.37-48
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• 2020

This research proposes how to enhance low customer satisfaction with health screening services caused by procedural complexity and limits of health screening. The purpose of this study is to identify sub-components of the service quality provided by general health examination centers. This is a qualitative analysis of in-depth interviews of providers and consumers of medical services. The data were primarily analyzed by affinity diagram, and the data were sorted and analyzed according to the criteria suggested by Donabedian's four components. Four types of quality factors and the health screening service quality components of 39 subordinate items were assessed. Components related to the use of IT facilities comprise a significant amount of the physical factors, and there are high demands for IT facilities among customers.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.3817-3825
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• 2015

Background: The human protein methyl-transferase DOT1L catalyzes the methylation of histone H3 on lysine 79 (H3K79) at homeobox genes and is also involved in a number of significant processes ranging from gene expression to DNA-damage response and cell cycle progression. Inhibition of DOT1L activity by shRNA or small-molecule inhibitors has been established to prevent proliferation of various MLL-rearranged leukemia cells in vitro, establishing DOT1L an attractive therapeutic target for mixed lineage leukemia (MLL). Most of the drugs currently in use for the MLL treatment are reported to have low efficacy, hence this study focused on various natural compounds which exhibit minimal toxic effects and high efficacy for the target receptor. Materials and Methods: Structures of human protein methyl-transferase DOT1L and natural compound databases were downloaded from various sources. Virtual screening, molecular docking, dynamics simulation and drug likeness studies were performed for those natural compounds to evaluate and analyze their anti-cancer activity. Results: The top five screened compounds possessing good binding affinity were identified as potential high affinity inhibitors against DOT1L's active site. The top ranking molecule amongst the screened ligands had a Glide g-score of -10.940 kcal/mol and Glide e-model score of -86.011 with 5 hydrogen bonds and 12 hydrophobic contacts. This ligand's behaviour also showed consistency during the simulation of protein-ligand complex for 20000 ps, which is indicative of its stability in the receptor pocket. Conclusions: The ligand obtained out of this screening study can be considered as a potential inhibitor for DOT1L and further can be treated as a lead for the drug designing pipeline.

• KSBB Journal
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• v.28 no.3
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• pp.185-190
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• 2013

For the selection of peptide specifically binding to $Pb^{2+}$, the biopanning with the commercially available Ph.D.-7 phage displayed heptapeptide library was carried out against $Pb^{2+}$ immobilized on a metal-chelating IDA (iminodiacetic acid) resin. After four rounds of screening against $Pb^{2+}$-IDA including negative selections against charged bead with metal ions other than $Pb^{2+}$ and uncharged bead, several candidate lead-binding phage peptides were initially determined based on the order of frequency from the screened phage clones. Of the selected phage peptide sequences, the peptide of the highest frequency, CysSerIleArgThrLeuHisGlnCys (CSIRTLHQC) also exhibited the strongest affinity for $Pb^{2+}$ in binding assays for individual phage clones. However, there was not a significant difference in $Pb^{2+}$ affinity between selected peptides when using synthetic heptapeptides corresponding to the displayed peptide sequences of phage clones.

• BMB Reports
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• v.39 no.1
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.