N dodecyl β d maltoside

LH2 protein was isolated from the cultured cells of a purple photosynthetic bacterium Phaeospirillum molischianum DSM120^{26 (link)}. BChl a was isolated from a purple photosynthetic bacterium Rhodobacter sphaeroides, and was converted to AcChl a and BPhe a by DDQ oxidation²⁷ and demetallation under acidic conditions^{28 (link)}, respectively. Lycopene and DDQ were purchased from Wako Pure Chemical Industries. A detergent n-dodecyl-β-D-maltoside (DDM) was purchased from Dojindo Laboratories.

The Escherichia coli strains, DH5α and BL21(DE3), were used as hosts for DNA manipulation and for protein expression, respectively. An expression plasmid of histidine-tagged RxR was constructed as previously described^{10 (link)} and RxR mutant genes were constructed by the SLiCE method^{30 (link)} using the gene of histidine-tagged RxR as a template. Consequently, these genes were inserted into pET21a (for wild-type RxR) or pET22b (for all RxR mutants) plasmid vectors (Novagen, USA) with NdeI and XhoI restriction enzyme sites. An expression plasmid of histidine-tagged Archaerhodopsin-3 (AR3) was constructed as previously described^{22 (link)}. E. coli cells harboring the plasmids were grown at 37 °C in LB medium (1% Bacto tryptone, 0.5% yeast extract and 0.5% NaCl) containing 50 μg/mL ampicillin and protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, final conc. = 1 mM) and all-trans retinal (final conc. = 10 μM). The cells were disrupted by sonication and crude membrane fractions were solubilized with n-dodecyl-β-D-maltoside (DDM, DOJINDO, Japan, 1.5 w/v %) and then purified by Ni²⁺ affinity column chromatography against the histidine-tag. Purified proteins were concentrated by centrifugation using an Amicon Ultra filter (30,000 MW cutoff; Merck Millipore, USA) and then replaced by the appropriate buffer solution.

E. coli BL21(DE3) was transformed with pCold-Pex11p and protein expression was induced at 15°C for 24 h with 0.1 mM isopropyl β-D-thiogalactoside (IPTG). Cells were harvested and lysed in sarcosine buffer 50 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 2 M Urea, 50 mM imidazole, 0.5% sodium N-lauroylsarcosine. After 15-min incubation on ice, the lysate was ultracentrifuged at 42,000 rpm for 30 min at 4°C using a Hitachi TLA100.3 rotor (Hitachi, Tokyo, Japan). The supernatant was incubated with Ni-NTA beads (Qiagen, Düsseldorf, Germany) at 4°C for 1 h, then the beads were collected and washed with wash buffer 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium N-lauroylsarcosine. Protein was eluted by washing buffer containing 0.3 M imidazole. The elution buffer was used for protein reconstitution in the control of the experiment. For purification of MBP-EGFP-Pex11p proteins, cells were lysed in buffer containing n-dodecyl-β-D-maltoside (Dojindo, Kumamoto, Japan), and protein was affinity-purified by amylose resin (New England BioLabs, Hertfordshire, UK) according to the manufacturer's instructions. We named MBP-EGFP-Pex11p protein as “EGFP-Pex11p” and used as such, since the MBP-tag was not readily cleaved off by PreScission protease (GH Healthcare, Little Chalfont, UK).