Purification of functional full-length liver X receptor β produced in Escherichia coli

Gudrun Toresson; Gertrud U. Schuster; Knut R. Steffensen; Martin Bengtsson; Jan Ljunggren; Karin Dahlman-Wright; Jan Åke Gustafsson

doi:10.1016/j.pep.2004.01.007

Purification of functional full-length liver X receptor β produced in Escherichia coli

Gudrun Toresson, Gertrud U. Schuster, Knut R. Steffensen, Martin Bengtsson, Jan Ljunggren, Karin Dahlman-Wright, Jan Åke Gustafsson

Houston Methodist

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

Liver X receptor β(LXRβ) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily. LXRβ and its isoform LXRα have recently been recognized as important regulators of lipid homeostasis in vertebrates. N-terminally hexahistidine-tagged rat LXRβ was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration. From 10 g of bacterial cells, 2.5 mg of protein was recovered. The purified LXRβ is functional with respect to ligand-, DNA-, and coactivator-binding. The synthetic ligand T0901317 bound to LXRβ with high affinity yielding a K_d of 2.7 nM. Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay. Furthermore, surface plasmon resonance analysis of LXRβ binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250. The purified LXRβ constitutes an important tool for further functional and structural studies.

Original language	English (US)
Pages (from-to)	190-198
Number of pages	9
Journal	Protein Expression and Purification
Volume	35
Issue number	2
DOIs	https://doi.org/10.1016/j.pep.2004.01.007
State	Published - Jun 2004

Keywords

Coactivator
Expression in E. coli
Hexahistidine tag
Liver X receptor
Nuclear receptor
RAP2050
Response element
Retinoid X receptor

ASJC Scopus subject areas

Biochemistry

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10.1016/j.pep.2004.01.007

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@article{cc2351ef88c341e8a92fa0125637aa28,

title = "Purification of functional full-length liver X receptor β produced in Escherichia coli",

abstract = "Liver X receptor β(LXRβ) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily. LXRβ and its isoform LXRα have recently been recognized as important regulators of lipid homeostasis in vertebrates. N-terminally hexahistidine-tagged rat LXRβ was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration. From 10 g of bacterial cells, 2.5 mg of protein was recovered. The purified LXRβ is functional with respect to ligand-, DNA-, and coactivator-binding. The synthetic ligand T0901317 bound to LXRβ with high affinity yielding a Kd of 2.7 nM. Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay. Furthermore, surface plasmon resonance analysis of LXRβ binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250. The purified LXRβ constitutes an important tool for further functional and structural studies.",

keywords = "Coactivator, Expression in E. coli, Hexahistidine tag, Liver X receptor, Nuclear receptor, RAP2050, Response element, Retinoid X receptor",

author = "Gudrun Toresson and Schuster, {Gertrud U.} and Steffensen, {Knut R.} and Martin Bengtsson and Jan Ljunggren and Karin Dahlman-Wright and Gustafsson, {Jan {\AA}ke}",

note = "Funding Information: We thank Thomas Kieselbach at the Protein Analysis Unit, Centre for Structural Biochemistry, Karolinska Institute, for mass spectrometry analysis and N-terminal amino acid sequencing and Dorothee Feltkamp for construction of the LXRβ expression plasmid. This work was supported by grants from the Swedish Research Council (31X-02819, 31X-06807), the Thuring, Tore Nilsson, and {\AA}lderssjukdomar foundations. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.",

year = "2004",

month = jun,

doi = "10.1016/j.pep.2004.01.007",

language = "English (US)",

volume = "35",

pages = "190--198",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "Academic Press",

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T1 - Purification of functional full-length liver X receptor β produced in Escherichia coli

AU - Toresson, Gudrun

AU - Schuster, Gertrud U.

AU - Steffensen, Knut R.

AU - Bengtsson, Martin

AU - Ljunggren, Jan

AU - Dahlman-Wright, Karin

AU - Gustafsson, Jan Åke

N1 - Funding Information: We thank Thomas Kieselbach at the Protein Analysis Unit, Centre for Structural Biochemistry, Karolinska Institute, for mass spectrometry analysis and N-terminal amino acid sequencing and Dorothee Feltkamp for construction of the LXRβ expression plasmid. This work was supported by grants from the Swedish Research Council (31X-02819, 31X-06807), the Thuring, Tore Nilsson, and Ålderssjukdomar foundations. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.

PY - 2004/6

Y1 - 2004/6

N2 - Liver X receptor β(LXRβ) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily. LXRβ and its isoform LXRα have recently been recognized as important regulators of lipid homeostasis in vertebrates. N-terminally hexahistidine-tagged rat LXRβ was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration. From 10 g of bacterial cells, 2.5 mg of protein was recovered. The purified LXRβ is functional with respect to ligand-, DNA-, and coactivator-binding. The synthetic ligand T0901317 bound to LXRβ with high affinity yielding a Kd of 2.7 nM. Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay. Furthermore, surface plasmon resonance analysis of LXRβ binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250. The purified LXRβ constitutes an important tool for further functional and structural studies.

AB - Liver X receptor β(LXRβ) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily. LXRβ and its isoform LXRα have recently been recognized as important regulators of lipid homeostasis in vertebrates. N-terminally hexahistidine-tagged rat LXRβ was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration. From 10 g of bacterial cells, 2.5 mg of protein was recovered. The purified LXRβ is functional with respect to ligand-, DNA-, and coactivator-binding. The synthetic ligand T0901317 bound to LXRβ with high affinity yielding a Kd of 2.7 nM. Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay. Furthermore, surface plasmon resonance analysis of LXRβ binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250. The purified LXRβ constitutes an important tool for further functional and structural studies.

KW - Coactivator

KW - Expression in E. coli

KW - Hexahistidine tag

KW - Liver X receptor

KW - Nuclear receptor

KW - RAP2050

KW - Response element

KW - Retinoid X receptor

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JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 2

ER -

Purification of functional full-length liver X receptor β produced in Escherichia coli

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Keywords

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