Expression, Antibodies Preparation And Functional Of EIF-5A

The eukaryotic initiation factor 5A (eIF-5A) is one of the most abundant initiation factors in the eukaryote, and it is the only protein in nature that contains a hypusine residue. At first, the gene of eIF-5A was fished from Mo-7e cell line using RT-PCR. Then we successfully construct the prokaryotic expression vectors of eIF-5A. After expression and purification, rheIF-5A was identified by western-blot and peptide mass-fingerprinting. Then protein crystal of eIF-5A was observed using atomic force microscopy. Further, the polyclonal and monoclonal antibodies were gained from immunized rabbits and rats. Hypusine-contained eIF-5A plays a role in cell growth and differentiation. We successfully construct the eukaryotic expression vector of GFP-eIF-5A and its mutational vector using genetic engineering techniques. We found that eEF-5 A localized in nucleus as well as in cytoplasm just for a short time after its transient expression, then distributed only in cytoplasm. It showed that eIF-5A subcellular localization was a dynamic process, and it might be a nucleocytoplasmic shuttle protein. Our study is the first to show that the inhibition of hypusine formation altered the subcellular distribution of GFP-eIF-5 A, and made it unable to localize at its proper site of action. Further, eIF-5A overexpression through transfection promoted UP inhibitor-induced apoptosis, and.mutation of eIF-5 A abolished the enhancing effect, we could conclude that the accumulation of hypusine-absent eIF-5A played an important role in the strong induction of apoptosis by UP inhibitors.