pMT3-RhoGC

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	Proteins were expressed transiently in HEK293-GnT1− cells by using calcium phosphate precipitation for transfection, and cells were harvested 72 h post-transfection. All-trans retinal (10 μm) was added to the cell suspension in the dark, followed by an incubation period of 30 min at 4 °C.

Protocol tips
Proteins were expressed transiently in HEK293-GnT1− cells by using calcium phosphate precipitation for transfection, and cells were harvested 72 h post-transfection. All-trans retinal (10 μm) was added to the cell suspension in the dark, followed by an incubation period of 30 min at 4 °C.

Publication protocol

"The gene for RhoGC was from BeGC1 (accession number KF309499) and had been codon-optimized for expression in E. coli (Genewiz Inc., South Plainfield, NJ). The gene was inserted between the EcoRI and NotI restriction sites of the mammalian expression vector pMT3 (20). Epitope tags were added at the N terminus (C8 epitope, placed between amino acids 1 and 2 of the native sequence) and C terminus (a GSGS linker followed by a T9A mutant of the 1D4 epitope was added after the last amino acid of the native sequence). Mutations were introduced by QuikChange II site-directed mutagenesis (Agilent).

The expression and purification of RhoGC and mutants were performed essentially as described previously for bovine rhodopsin (20). Proteins were expressed transiently in HEK293-GnT1− cells by using calcium phosphate precipitation for transfection, and cells were harvested 72 h post-transfection. All-trans retinal (10 μm) was added to the cell suspension in the dark, followed by an incubation period of 30 min at 4 °C. All procedures following the addition of retinal were performed in the dark under a dim red light. Cells were lysed in 2% DDM or DM detergent for 1 h at 4 °C. The lysate was centrifuged at 3500 rpm and 4 °C for 10 min to remove nuclei, and the post-nuclear supernatant fraction was applied to an immunoaffinity matrix (either anti-C8 or anti-1D4). The proteins were allowed to bind to the column for 2 h at 4 °C before the non-bound fraction was removed. The column was then washed at 4 °C with 0.1% (w/v) detergent (DM or DDM) in PBS. Purified protein was eluted at room temperature with 80 μm peptide (C8 or 1D4)."

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