pNIC28‐Bsa4-bops

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	Positive clones were transformed into E. coli C43‐Rosetta BL21 (DE3) cells for protein expression. Protein expression was induced at A600 ~ 0.6 OD with 0.5 mM IPTG. The culture was supplemented with 5–10 μM retinal to enhance BR maturation and functional expression.

Protocol tips
Positive clones were transformed into E. coli C43‐Rosetta BL21 (DE3) cells for protein expression. Protein expression was induced at A600 ~ 0.6 OD with 0.5 mM IPTG. The culture was supplemented with 5–10 μM retinal to enhance BR maturation and functional expression.

Publication protocol

The amplified bops were cloned in pNIC28‐Bsa4 and pET22b between the Nhe‐XhoI restriction sites for expression as C‐terminal 6× His‐tagged proteins or the pET28a vector (modified) between the NdeI‐XhoI sites for expression as a Mistic fusion protein. Positive clones were transformed into E. coli C43‐Rosetta BL21 (DE3) cells for protein expression. Protein expression was induced at A600 ~ 0.6 OD with 0.5 mM IPTG. The culture was supplemented with 5–10 μM retinal to enhance BR maturation and functional expression. Trans‐retinal was purchased from Sigma‐Aldrich, (St. Louis, MO, USA) (R2500‐1G), and stocks were prepared in 100% ethanol. The culture was incubated at 37°C and 200 rpm for 5 h in an incubator shaker. The cells were harvested at 9000 × g for 10 min and resuspended in lysis buffer A (50 mM Tris and 150 mM NaCl, pH 8.0). The cells were lysed by sonication, and the insoluble membrane fraction was obtained by centrifuging the lysate at high speed at 18 000 × g for 30 min. The soluble fraction was discarded, and the pellet was resuspended in lysis buffer B (50 mM Tris and 150 mM NaCl pH 8 with 0.2% DDM). For proteins expressed with the Mistic tag, 0.2% SDS detergent was used instead of DDM. The resuspended insoluble fraction was incubated overnight with gentle shaking. DDM and SDS were used to facilitate BR extraction from the insoluble lipid bilayer, thus solubilizing the protein. The soluble fraction of the protein was mixed with Ni‐NTA resin for binding, and the proteins were eluted using elution buffer E (50 mM Tris and 150 mM NaCl, pH 8.0 with 0.02% DDM or 0.02% SDS in the case of the Mistic tag with 500 mM imidazole).

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