Purification of SAR11 HIMB114 TAT Rhodopsin

The gene of SAR11 HIMB114 TAT rhodopsin, whose codon was optimized for an Escherichia coli expression system, was synthesized (Eurofins Genomics Inc.) and subcloned into a pET21a (+)-vector with a C-terminal 6×His-tag [21 (link),22 (link)]. For mutagenesis, a Quick-change site-directed mutagenesis kit (Stratagene) was used based on a standard protocol [23 (link),24 (link)]. WT and T82D mutant protein were expressed in the E. coli C43 (DE3) strain. The plasmid was prepared using the Nucleo Spin Plasmid Easy pure kit and 3 mL bacterial culture initiated from single colonies selected from a transformation plate. Protein expression was induced by 1.0 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 4 h at 37°C in the presence of 10 μM all-trans-retinal (Sigma-Aldrich). The expressed proteins were purified from E. coli cells according to previously reported methods [23 (link),24 (link)]. The cells were disrupted with a French Press (Ohtake) and the membrane fraction was collected by ultracentrifugation at 125,000 g for 1 h. The protein was solubilized in 2.0% n-dodecyl-β-D-maltoside (DDM) in the presence of 300 mM NaCl, 5 mM imidazole and 50 mM MES (pH 6.5). After Co²⁺-NTA affinity chromatography, the collected fractions were dialyzed against a buffer containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl and 0.03 % DDM to remove imidazole.