Research On The Expression Purification And Interactions Of Adiponectin And Its Receptor

Adiponectin, secreted by Adipose tissue, can identify and intergrate Adip R1/2 in the liver and skeletal muscle, stimulate glucose sugar intake and fatty acid oxidation through AMPK/ PPARα signaling pathway, maintain energy homeostasis, play insulin-sensitizating function.Abnormal plasma levels and physiological function of adiponectin and its receptors will lead a series of diseases,including insulin resistance,obesity,atherosclerosis and so on. Condition will have a alleviate by recoving the its plasma levels and physiological function.Therefore,adiponectin and its receptors will be a novel treatment modifity for insulin resistance..However, the details of interactions between adiponcetin and Adip R1 is unknow.We purify adiponectin and Adip R1 protein by constructing recombinant protein expression system in vitro.For Adiponectin,we construct prokaryotic and eukaryotic expression system,identifiy and optimize expression chone.We affinity purify Adiponectin-GST and Adiponectin-MBP protein.We proteolysis GST or MBP fusion protein by PPase or TEV protease. Size-exclusion chromatography results of Adiponectin indicate Adiponectin can form dimers.For Adip R1,we design three truncations,Adip R1(22-375) 、 Adip R1(72-375) 、 Adip R1(82-375) 。 We optimize expression and purification conditions,affinity purify three truncation Adip R1 protein. Size-exclusion chromatography results of Adiponectin indicate it can form dimers by disulfide donds.On this basis,we study their interactions by pull down and size-exclusion chromatography experiment. Results showe adiponectin and Adip R1 have no obvious interactions in vitro.