An application of fast protein liquid chromatography (FPLC) in the purification of retinol binding protein from rat serum

Summary Retinol Binding Protein (RBP) is the specific plasma protein for the transport of retinol from liver to peripheral tissues. It is a single polypeptide chain of approximately 21 KDa, and circulates as a 11 molar complex with transthyretin (TTR). The relative low concentration in plasma (40–50 g/ml and its chromatographic behaviour on ionic exchangers render the purification of rat RBP particularly laborious. In this paper we report a simple and semi-automatic method for the preparative purification to homogeneity of rat serum RBP. The method includes: (1) Selective removal of albumin by affinity chromatography on a Blue Sepharose column; (2) Chromatography on a Mono Q strong anion exchange column; (3) Dissociation of the RBP-TTR complex by 3 M urea; (4) Concentration, desalting and freeze drying. The purified RBP has been used for the production in rabbit of antirat RBP specific antibodies for studies on nutritional control of RBP synthesis and metabolism.