Expression and Purification of Recombinant Human RBP4

Human RBP4 expression and purification from Escherichia coli was accomplished essentially as described previously (Shi et al., 2017 (link)). Briefly, human RBP4 (hRBP4) cDNA was cloned into a pET3a expression vector and expressed in BL-21 DE3 cells according to a standard protocol. Bacterial cells were harvested and lysed by osmotic shock. Insoluble material was pelleted by centrifugation, washed, and solubilized in 7M guanidine hydrochloride and 10 mM dithiothreitol. After overnight incubation, insoluble material was removed by ultracentrifugation, and the supernatant was used for the hRBP4 refolding procedure. hRBP4 was refolded by the dropwise addition of solubilized material into a mixture containing 150 μCi of [11,12-³H]ROL ([³H]ROL) (PerkinElmer Life Sciences) and non-radiolabeled ROL (Sigma) at a final concentration of 1 mm. Refolded holo-hRBP4 was dialyzed against 10 mM Tris/HCl buffer, pH 8.0, and loaded onto a DE53 anion exchange chromatography column (Whatman, Piscataway, NJ). Holo-hRBP4 was eluted with linear gradient of NaCl (0–1M) in 10 mM Tris/HCl buffer, pH 8.0. Collected fractions were examined by SDS-PAGE and UV-visible spectroscopy to ensure a proper protein/retinoid ratio. Fractions containing at least 90% holo-hRBP4 were pooled together and concentrated in a Centricon centrifugal filter device (cut-off 10,000 Da) (Millipore, Billerica, MA) to 5 mg/mL. [³H]ROL was quantified in a scintillation counter (Beckman Coulter, Indianapolis, IN). Holo-hRBP4 aliquots were stored at −80°C until used.