Isolation And Purification Of Bovine Lactoferrin Receptor And Study On Its Binding Properties

Lactoferrin?Lf?,a kind of iron-binding glycoprotein with multiple biological functions,widely present in mammalian milk and many other organizations and their secretions.And bovine lactoferrin?bLf?as a functional nutrition protein has been widely applied to food,medicine and other fields.Lf receptors?LfR?expressed in various types of mammalian cells are considered to be responsible for mediating some of the functions of Lf.The gut is the main place of Lf functions.Therefore,the study of intestinal lactoferrin receptors will help to further elucidate on the mechanisms of biological function of Lf.3-4 weeks old BALB / C mice was chosen as test subjects.To isolated mouse intestinal epithelial cells?IEC?,two kinds of enzymes digestion protocols were used to identify the suitable method.After adjusting the cell concentration,the IEC plasma membranes was isolated by differential centrifugation,and plasma membrane proteins was further purified with detergent CHAPS and ultrafiltration and divided into groups according to molecular weight.We applied ForteBio's Octet RED 96 system to measure the interaction of different components of membrane proteins with bLf,and analysed its influensing factors.The results show that the mammalian small intestinal mucosa may be express a new bLf receptor,the molecular weight is30-100 KD,and the best binding pH was in neutral condition,Ca²⁺enhanced the binding of lactoferrin and intestinal mucosa receptor,but the effect is very weak.We applied AminoLink Plus affinity chromatography to purify intestinal bLf receptor,the purity of protein was identified by capillary electrophoresis.We use molecular inieraction systrem to measure affinity constant between bLf and its intestinal mucosa receptor,and the molecular weight of bLf receptor was identified by Q-TOF LC/MC.Capillary electrophoresis showed that the receptor purified by affinity chromatography has only one characteristic peak,indicating that there is only a component in blf receptor protein.The dynamics analysis showed that the receptor has a high degree of specific binding properties with blf,its binding constant?KD?is4.38E-10,and the molecular weight of the receptor is aboult 80 KD.These result suggest that the receptor isolated from mice small intestine mucosa is a new bLf specific recptor.