[Molecular cloning and identification and biological activity of GIF-interacting protein(s)]

In order to examine the elusive functional mechanism of GIF (Neuronal growth inhibitory factor, GIF) and elucidate the possible relationship between GIF and Alzheimer's disease, we constructed bait1 plasmid (pHyblex-GIF) by cloning GIF cDNA directly in frame with plasmid pHyblex, and used the yeast two-hybrid system to screen Alzheimer's disease human brain cDNA library and found the GIF-interacting proteins. The final results from coimmunoprecipitation and western blotting experiments confirmed that interacting proteins specifically binds to GIF. After sequencing the nucleotide of the putative positive plasmids and searching for homologues, we found that one of these is the part of human nuclear dUTPase protein sequence. Then the dUTPase genes are cloned into pGEX-4T-1, the fusion expression vector of GST,and highly expressed in E. coli BL21. The proteins dUTPase and GIF were purified and obtained by affinity chromatography, thrombin digestion and gel filtration on Sephacryl S100. It demonstrated that the proteins dUTPase and GIF had the growth inhibitory activity on co-cultured neuron in vitro. The inhibitory curve was very similar to the GIF. It's possible that dUTPase is one of the proteins interacting with GIF in Alzheimer's disease human brain extracts.