Purification of GPCR Samples for Structural Studies

Purified GPCR samples (β₂AR, A_2AAR, glucagon receptor, and 5-HT_2B)^{24 (link),28 (link)–31 (link)} were prepared as follows. Constructs engineered for crystallization were expressed in Sf9 insect cells for 48 h at 27 °C using recombinant baculovirus. Cells were harvested and total membranes were purified by repeated Dounce homogenization and centrifugation in hypotonic and hypertonic buffer. GPCR-ligand complexes were subsequently formed by incubating purified membranes in the presence of ligand, followed by extraction of the complexes in 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) and 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma). Solubilized proteins were purified by immobilized metal affinity chromatography (IMAC) and concentrated to ~20 – 50 mg/mL.

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Weierstall U., James D., Wang C., White T.A., Wang D., Liu W., Spence J.C., Doak R.B., Nelson G., Fromme P., Fromme R., Grotjohann I., Kupitz C., Zatsepin N.A., Liu H., Basu S., Wacker D., Han G.W., Katritch V., Boutet S., Messerschmidt M., Williams G.J., Koglin J.E., Seibert M.M., Klinker M., Gati C., Shoeman R.L., Barty A., Chapman H.N., Kirian R.A., Beyerlein K.R., Stevens R.C., Li D., Shah S.T., Howe N., Caffrey M, & Cherezov V. (2014). Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nature communications, 5, 3309.

Publication 2014

n-dodecyl-β-D-maltopyranoside (DDM, cholesteryl hemisuccinate (CHS,

Affinity chromatography Baculovirus Buffer Cells Centrifugation Cholesteryl hemisuccinate Crystallization Glucagon receptor Insect Ligand Membranes Metal Proteins Sf9 cells

Corresponding Organization : Arizona State University

Other organizations : Scripps Research Institute, Center for Free-Electron Laser Science, Universität Hamburg, SLAC National Accelerator Laboratory, Menlo School, Max Planck Institute for Medical Research, Trinity College Dublin

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Variable analysis

independent variables

Purified GPCR samples (β2AR, A2AAR, glucagon receptor, and 5-HT2B)

dependent variables

Not explicitly mentioned

control variables

Constructs engineered for crystallization
Expression in Sf9 insect cells for 48 h at 27 °C using recombinant baculovirus
Harvesting of cells and purification of total membranes by repeated Dounce homogenization and centrifugation in hypotonic and hypertonic buffer
Incubation of purified membranes in the presence of ligand
Extraction of GPCR-ligand complexes in 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) and 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma)
Purification of solubilized proteins by immobilized metal affinity chromatography (IMAC)
Concentration of purified proteins to ~20 – 50 mg/mL

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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