The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase

Moaddel, Ruin; Price, Gerry B.; Juteau, Jean-Marc; Leffak, Michael; Wainer, Irving W.

doi:10.1016/j.jchromb.2005.03.030

Cited by 4 publications

(7 citation statements)

References 15 publications

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“…We have previously demonstrated that columns which contain immobilized nuclear proteins, the estrogen receptor (ER) ligand binding domain [7] and the DNA unwinding element binding (DUE-B) protein [8], can be created, characterized and used to study ligand-protein interactions. In these studies, the ERRα and ERRγ ligand binding domains were covalently immobilized via the N-terminus onto the surface of an aminopropyl silica liquid chromatography stationary to create the ERRα-silica and ERRγ-silica columns, or on the activated surface of open tubular glass capillaries to create the ERRα-OT and ERRγ–OT columns.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and characterization of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor α and estrogen related receptor γ

Sanghvi¹,

Moaddel²,

Frazier³

et al. 2010

Journal of Pharmaceutical and Biomedical Analysis

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The ligand binding domains of the estrogen related receptors, ERRα and ERRγ were covalently immobilized onto the surface of an aminopropyl silica liquid chromatography stationary phase to create the ERRα-silica and ERRγ-silica columns and onto the surface of open tubular capillaries to create the ERRα-OT and ERRγ-OT columns. The ERR-silica and ERR-OT columns were characterized using frontal chromatographic techniques with diethylstibesterol and the binding affinities, Kd values, to the immobilized receptors were consistent with the values obtained by a radioligand binding assay. The ERRγ-silica column was also characterized using non-linear chromatographic techniques using a series of tamoxifen derivatives. The relative Kd values obtained for the derivatives were consistent with the relative ability of the compounds to inhibit the cellular proliferation of the human-derived T98G glioma cell line, expressed as IC50 values. The results indicate that the columns containing immobilized ERRα and ERRγ can be created and used to characterize the binding of compounds to the immobilized receptors and that the relative retention of compounds on these columns reflects the magnitude of their inhibitory activity.

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Section: Introductionmentioning

confidence: 99%

Synthesis and characterization of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor α and estrogen related receptor γ

Sanghvi¹,

Moaddel²,

Frazier³

et al. 2010

Journal of Pharmaceutical and Biomedical Analysis

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“…It has been previously demonstrated that His-tagged fusion protein could be immobilized using a Nickel coated stationary phase and used for characterization of the fusion protein [45, 47]. In the current study, they demonstrated that the calmodulin-melittin complex was screened against 50 mixtures of 20 compounds and correctly identified three known antagonists as well as 2 previously unknown antagonist.…”

Section: Application Of Biochromatographymentioning

confidence: 53%

“…Directional immobilization can be carried out using various techniques, including the use of fusion proteins with either a His-tag [45] or glutathione S-transferase (GST) tagged fusion proteins [46]. In addition, the proteins can be immobilized with their corresponding binder, nickel and glutathione, respectively.…”

Section: Experimental Approachmentioning

confidence: 99%

“…In addition, the proteins can be immobilized with their corresponding binder, nickel and glutathione, respectively. This approach (His-tag) has been used to immobilize the His-tagged estrogen receptor ligand binding domain, ER-LBD [47] and the His-tagged DNA unwinding element binding (DUE-B) [45] using Ni +2 as the coordinating metal ion. In addition Jonker et al, used a Cobalt (II) coated magnetic bead to immobilize the His-tagged protein [32], while McFadden et al, used a Nickel coated magnetic bead to isolate a His-tagged protein from a mixture of proteins and small molecules [31].…”

Section: Experimental Approachmentioning

confidence: 99%

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The development and characterization of protein-based stationary phases for studying drug–protein and protein–protein interactions

Sanghvi

Moaddel

Wainer

2011

Journal of Chromatography A

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Protein-based liquid chromatography stationary phases are used in bioaffinity chromatography for studying drug-protein interactions, the determination of binding affinities, competitive and allosteric interactions, as well as for studying protein-protein interactions. This review addresses the development and characterization of protein-based stationary phase, and the application of these phases using frontal and zonal chromatography techniques. The approach will be illustrated using immobilized heat shock protein 90 and the immobilized estrogen related receptor stationary phases. In addition, the review discusses the use of the protein-coated magnetic beads for ligand and protein fishing as well as for the identification of unknown ligands from cellular or botanical extracts.

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“…Unlike classical zonal elution technique, the small ligand is only playing a role of the interaction mediator between the two proteins, one of which is immobilized on a solid support, and another is flowed over it. This modification of the method, where a ligand mediates the interaction between two or more proteins, has already been utilized; − however, those protein/ligand/protein interactions were long-lived enough to allow “fishing” out of interacting proteins or, in the other case, is a long-lived DNA/ligand/protein interaction that was assessed for function upon different ligand binding. Here, the ligand binding allows for a very transient interaction that cannot be detected by any other method.…”

mentioning

confidence: 99%

Modification of the Zonal Elution Method for Detection of Transient Protein–Protein Interactions Involving Ligand Exchange

Sjoelund

Kaltashov

2012

Anal. Chem.

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A new affinity chromatography method was developed by modifying a zonal elution method. The new method targets transient protein-protein interactions, such as those encountered during direct ligand transfer between the ligand transporter and its cognate receptor. A ligand-loaded transport protein is immobilized on the solid support, and a plug containing a putative receptor is flowed through the column. Elution profiles of proteins not interacting with the immobilized transporter can be approximated with a simple Gaussian curve, while the elution profiles of cognate receptors show significant delay and exhibit complex shape. Ligand transfer from the immobilized transporter molecules to the receptors is verified by both UV absorbance measurements and mass spectrometry. The sensitivity of the method is demonstrated using retinoic acid (RA) transfer from various isoforms of cellular RA binding proteins (CRABPs) and RA receptor γ (RARγ). Although these interactions have been hypothesized long ago to proceed via direct mechanism (i.e., via transient docking of the receptor and the transporter), the existing biophysical techniques failed to detect the presence of the transporter-receptor complexes. However, the modified zonal elution method provides unequivocal evidence of direct interaction between RARγ and one of the CRABP isoforms (CRABP II) during the ligand transfer to the receptor.

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The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase

Cited by 4 publications

References 15 publications

Synthesis and characterization of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor α and estrogen related receptor γ

Synthesis and characterization of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor α and estrogen related receptor γ

The development and characterization of protein-based stationary phases for studying drug–protein and protein–protein interactions

Modification of the Zonal Elution Method for Detection of Transient Protein–Protein Interactions Involving Ligand Exchange

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