2.2. Expression and Purification of CRABP1 and CRABP2

CRABP1 and CRABP2 expression constructs were a gift from Noa Noy (Case Western Reserve University). These CRABP1 and CRABP2 clones, in a pET28a vector, contained a thrombin cleavage site and an N-terminal hexa-histidine (6×his) tag. CRABP1 contained an additional N-terminal T7 tag (Figure S1) The plasmid map and sequences for CRABP1 and CRABP2 are shown in Supplementary Materials. Both proteins were expressed in Rosetta 2 E. coli (Novagen, Madison, WI). A 25 mL starter culture with a freshly transformed colony in LB-kanamycin (50 μg/mL) was grown for 6 h at 37 °C in a shaking incubator at 250 RPM. A total of 15 mL of the starter culture was used to inoculate 1 L of LB-kanamycin which was grown to an OD600 ≈ 0.6. The culture was cooled to room temperature over 20 min prior to adding 0.1 mM IPTG to induce CRABP1 and CRABP2 expression. The proteins were expressed at 18 °C for 18 h in a shaking incubator at 220 RPM. Cells were harvested by centrifugation at 5000 g for 20 min, washed with PBS containing 1 mM PMSF, pelleted, decanted and stored at −80 °C.

Frozen pellets were thawed on ice in 25 mL of lysis buffer per one L of culture (20 mM Tris pH 7.4, 500 mM NaCl and 30 mM imidazole) with protease inhibitor cocktail (Roche, cOmplete Mini EDTA-free), 1 mM PMSF and 25 U of benzonase. A total of 1 mg/mL lysozyme was added to the resuspended cells and the suspension was rocked on ice for 20 min. To ensure complete lysis, cells were sonicated at 75% power for 30 s with a 1 min rest on ice (5 rounds). The lysate was cleared via centrifugation at 20,000 g for 30 min and supernatant filtered through a 0.45 μm Amicon syringe filter (Milipore-Sigma, St. Louis, MO, USA). The filtered lysate was loaded onto a 90 mL Dynaloop (Biorad, Hercules, CA, USA) coupled to a DuoFlow fast protein liquid chromatography (FPLC) (Bio-Rad, Hercules, CA, USA) and a 1 mL HisTrap affinity column (GE Healthcare, Chicago, IL, USA), equilibrated in lysis buffer at flow rate of 15 mL/min. For protein purification, the column was washed with 10 column volumes of wash buffer (20 mM Tris pH 7.4, 500 mM NaCl, 30 mM imidazole) and CRABPs were eluted with 300 mM imidazole in elution buffer (20 mM Tris pH 7.4, 500 mM NaCl) in 1 mL fractions over 10 column volumes. Protein elution was detected at 280 nm absorbance and the CRABP-containing fractions combined. The eluted protein was concentrated, and buffer exchanged into thrombin cleavage buffer (20 mM Tris, pH 7.4, 500 mM NaCl) using a 10 kDa molecular weight cutoff (MWCO) Amicon concentrator (Milipore-Sigma, St. Louis, MO, USA). After buffer exchange, the protein concentration was measured by Nanodrop (Thermo Fisher, Waltham, MA, USA) by absorbance at 280 nm. The N-terminal 6×his tag was cleaved by adding 0.03 U of thrombin protease per 10 μg of CRABP into the solution and incubating overnight (>12 h) at 4 °C. Cleavage of the N-terminal tag was confirmed via SDS-PAGE with Coomassie staining and an anti-his (mouse anti-His antibody from Qiagen, Valencia, CA, USA) Western blot. The cleaved protein was then injected into a Superdex 75 size exclusion column (GE Healthcare, Chicago, IL, USA) equilibrated with HEK buffer (10 mM Hepes, pH 8, 100 mM KCl, 0.1 mM EDTA) using DuoFlow FPLC at a flow rate of 0.5 mL/min. After injection, the flow rate of the HEK buffer was increased to 1 mL/min and the CRABP-containing fractions, as detected at 280 nm, were collected, pooled and concentrated using a 10 kDa MWCO Amicon concentrator. The final concentration of CRABPs was quantified by a BCA protein assay. DTT (0.5 mM final concentration) and glycerol (50% final concentration) were added to the protein solution in HEK buffer and the CRABPs were stored at −20 °C. The binding of atRA to the CRABPs was verified via fluorescence titrations, as previously described [23]. Briefly, titrations were prepared in a 96-well black-walled plate with 2 µM CRABP and 0–2.8 µM atRA in 100 mM potassium phosphate, pH 7 under protection from light. The quenching of CRABP tryptophan fluorescence (excitation at 290 nm and emission at 340 nm) by atRA binding was measured at 37 °C with a Gemini Em fluorescence plate reader (Molecular Devices, San Jose, CA, USA) with the emission auto-cutoff set to on. Endpoint fluorescence scans were carried out with the photomultiplier tube (PMT) sensitivity set to auto with 6 reads and auto-calibration and auto-mixing for 2 s.