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Baculovirus method

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The Baculovirus method is a technique used for the expression and production of recombinant proteins. It utilizes a virus that specifically infects insect cells as the vector to deliver and express the target gene of interest. This method allows for the efficient production of complex proteins, making it a valuable tool in various research and industrial applications.

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13 protocols using Baculovirus method

Single chain construct of Fab16 (scFv16) was expressed and purified as previously described19 (link). In brief, a C-terminal 6× histidine tagged scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells using the baculovirus method (Expression Systems), and purified by Ni-NTA (Qiagen) chromatography. Supernatant from baculovirus-infected cells was pH balanced by addition of Tris pH 8.0. The C-terminal 6x His-tag of Ni-NTA eluent was cleaved by 3C protease, and the protein was dialyzed into a buffer consisting of 20 mM HEPES pH 7.5 and 100 mM NaCl. The sample was reloaded onto the Ni-NTA column to capture the cleaved His6. The flow-through containing scFv16 was collected, concentrated and purified through gel-filtration chromatography in a final buffer (100 mM NaCl and 20 mM HEPES pH 7.5). Monomeric fractions were pooled, concentrated to ~100 mg/ml.
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Single chain construct of Fab16 (scFv16) was expressed and purified as previously described19 (link). In brief, a C-terminal 6× histidine tagged scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells using the baculovirus method (Expression Systems), and purified by Ni-NTA (Qiagen) chromatography. Supernatant from baculovirus-infected cells was pH balanced by addition of Tris pH 8.0. The C-terminal 6x His-tag of Ni-NTA eluent was cleaved by 3C protease, and the protein was dialyzed into a buffer consisting of 20 mM HEPES pH 7.5 and 100 mM NaCl. The sample was reloaded onto the Ni-NTA column to capture the cleaved His6. The flow-through containing scFv16 was collected, concentrated and purified through gel-filtration chromatography in a final buffer (100 mM NaCl and 20 mM HEPES pH 7.5). Monomeric fractions were pooled, concentrated to ~100 mg/ml.
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Human full-length CB1 with N-terminal FLAG and C-terminal hexahistadine tag was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus method (Expression Systems). The same construct with an eGFP (CB1-eGFP) at the C-terminus was used for small-scale coupling and FSEC studies. The receptor was extracted from insect cell membranes with 1% lauryl maltose neopentyl glycol (L-MNG) and purified by nickel-chelating sepharose chromatography. The Ni-NTA pure eluate was applied to a Ml anti-FLAG immunoaffinity resin and washed with progressively decreasing concentration of inverse agonist, SR and increasing concentration of agonist FUB. The receptor was eluted in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% L-MNG, 0.005% cholesterol hemisuccinate (CHS), 2 μM FUB, FLAG peptide and 5 mM EDTA. The final step of purification was size exclusion chromatography on Superdex 200 10/300 gel filtration column (GE) in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.02% L-MNG, 0.002% CHS, and 2 μM FUB. Finally, agonist-bound CB1 was concentrated to ~500 μM and flash frozen and stored in −80 °C.
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Human 5-HT1A, 5-HT1D, and 5-HT1e, human DNGαi1, human Gβ1, and human Gγ2 were co-expressed in Spodoptera frugiperda Sf9 insect cells (Invitrogen) using the baculovirus method (Expression Systems). For the 5-HT1D–Gi and 5-HT1e–Gi complexes, scFv16 was co-expressed to stabilize the protein. As for the apo–5-HT1A–Gi (WT) complex used for GTPase assay, the DNGαi1 was replaced by WTGαi1. Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 2-3 million cells per ml and then infected with four separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection and cell pellets were stored at -80°C.
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Human FP, Gq chimera, Gβ1, Gγ, and scFv16 were co-expressed in High Five insect cells (Invitrogen) using the baculovirus method (Expression Systems). Cell cultures were grown in ESF 921serum-free medium (Expression Systems) to a density of 2–3 million cells per mL and then infected with six separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection, and cell pellets were stored at −80 °C.
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CB1 was expressed and purified as described previously13 (link). Briefly, human full-length CB1 containing an N-terminal FLAG tag and C-terminal histidine tag was expressed in Spodoptera frugiperda Sf9 insect cells with the baculovirus method (Expression Systems). Receptor was extracted using 1% lauryl maltose neopentyl glycol (L-MNG) and purified by nickel-chelating Sepharose chromatography. The eluant from the Ni column was applied to an M1 anti-FLAG immunoaffinity resin. After washing to progressively decreasing the concentration of L-MNG, the receptor was eluted in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% L-MNG, 0.005% cholesterol hemisuccinate (CHS), FLAG peptide and 5 mM EDTA. Finally, CB1 was purified with size exclusion chromatography, on Superdex 200 10/300 gel filtration column (GE) in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.02% L-MNG, 0.002% CHS. Ligand-free CB1 was concentrated to ~500 µM and stored at −80 °C.
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Human NMUR1, NMUR2, Gq chimera, Gβ1, Gγ, scFv16, and Ric8a were co-expressed in High Five insect cells (Invitrogen) using the baculovirus method (Expression Systems). Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 2-3 million cells per mL and then infected with six separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection, and cell pellets were stored at −80°C.
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Human IP, miniGαs, Gβ, and Gγ were coexpressed in High Five cells (Hi5, Invitrogen) using the baculovirus method (Expression Systems). Cells were grown in ESF 921 serum-free medium (Expression Systems) at 27°C and 120 rpm to a density of approximately two to three million cells/ml. Cells were then infected with different baculoviruses at a suitable ratio. At 48 hours postinfection, cells were harvested by centrifugation and stored at −80°C.
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β2AR was expressed in Sf9 insect cells using the baculovirus method (Expression Systems) and purified as described previously (50 (link)).
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5-HT1eR, Gγ2-Gαi1, and Gβ1 were expressed in Spodoptera frugiperda (Sf9) insect cells (Expression Systems) using the baculovirus method (Expression Systems). For the mianserin–5-HT1eR–Gi1 complex, receptor and heterotrimeric G protein were expressed and purified separately and assembled subsequently. For the setiptiline–5-HT1eR–Gi1 complex, receptor and G protein components were coexpressed, and scFv16 was added after complex purification to stabilize the signaling complex. Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 2 to 3 million cells/ml and then infected with separate baculoviruses either at a multiplicity of infection of 3 (receptor alone) and 2:2 (Gγ2-Gαi1:Gβ1) in the case of the mianserin complex or at a ratio of 2:1:1 (5-HT1eR:Gγ2-Gαi1:Gβ1) for the setiptiline complex. The culture was collected by centrifugation 48 hours after infection, and cell pellets were stored at −80°C until further use.
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