Heterologous Production of Active Mammalian G‐Protein‐Coupled Receptors Using Baculovirus‐Infected Insect Cells

Chiu, Mark L.; Estvander, Brian R.; Esbenshade, Timothy A.; Kakavas, S.J.; Krueger, Kathleen M.; Lake, Marc; Pereda‐Lopez, Ana

doi:10.1002/9783527634521.ch4

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“…The commonly used expression systems for GPCR include baculovirus-infected insect cells, yeast, bacterial or mammalian cells as well as cell-free systems [ 6 ]. Expression in insect cells has been particularly useful for production of receptors in milligram quantities for crystallization trials [ 5 , 9 , 10 , 13 , 14 ]. However, the adaptation of insect- or mammalian cells for preparation of stable-isotope labeled proteins for nuclear magnetic resonance (NMR) spectroscopy studies currently is prohibitively expensive due to the complexity of the medium and high cost of labeled nutrients [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Methods for Recombinant Expression and Functional Characterization of Human Cannabinoid Receptor Cb2

Yeliseev

2013

Computational and Structural Biotechnology Journal

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Cannabinoid receptor CB2 is a seven transmembrane-domain integral membrane protein that belongs to a large superfamily of G protein-coupled receptors (GPCR). CB2 is a part of the endocannabinoid system that plays vital role in regulation of immune response, inflammation, pain sensitivity, obesity and other physiological responses. Information about the structure and mechanisms of functioning of this receptor in cell membranes is essential for the rational development of specific pharmaceuticals. Here we review the methodology for recombinant expression, purification, stabilization and biochemical characterization of CB2 suitable for preparation of multi-milligram quantities of functionally active receptor. The biotechnological protocols include expression of the recombinant CB2 in E. coli cells as a fusion with the maltose binding protein, stabilization with a high affinity ligand and a derivative of cholesterol in detergent micelles, efficient purification by tandem affinity chromatography, and reconstitution of the receptor into lipid bilayers. The purified recombinant CB2 receptor is amenable to functional and structural studies including nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.

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Section: Introductionmentioning

confidence: 99%

Methods for Recombinant Expression and Functional Characterization of Human Cannabinoid Receptor Cb2

Yeliseev

2013

Computational and Structural Biotechnology Journal

View full text Add to dashboard Cite

show abstract

Heterologous Production of Active Mammalian G‐Protein‐Coupled Receptors Using Baculovirus‐Infected Insect Cells

Cited by 1 publication

References 139 publications

Methods for Recombinant Expression and Functional Characterization of Human Cannabinoid Receptor Cb2

Methods for Recombinant Expression and Functional Characterization of Human Cannabinoid Receptor Cb2

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