Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization

Human retinol-binding protein (RBP) is a monomeric 21-kDa protein that is currently the subject of numerous studies owing to its role in the cellular uptake and utilization of retinol. When the REP gene is overexpressed in Escherichia coli, inclusion bodies of aggregated REP are found in the cells. These inclusion bodies are solubilized in 5.0 M GdmCl containing 10 mM DTT. Refolding of REP is carried out in the presence of vitamin A by diluting denatured and reduced REP into a redox refolding buffer consisting of 3 mM cysteine/0.3 mM cystine at 4 degrees C. Ion exchange chromatography (HPLC) is utilized to purify refolded REP to homogeneity as demonstrated by SDS-PAGE and electrospray MS. The native structure of refolded REP was established by its ability to bind to vitamin A and the plasma protein transthyretin. The reconstitution of REP outlined within affords a 50-60% overall yield, i.e., 73 mg of pure RBP/L of E. coli culture. (C) 1998 Academic Press.