An in vitro study of the expression purification and intramolecular interactions of Hepatoma Up Regulated Protein (HURP)

Bachelor Thesis from the year 2011 in the subject Biology - Micro- and Molecular Biology grade: A+ or 10.0 Democritus University Of Thrace (Molecular Biology and Genetics) language: Greek Modern (1453-) abstract: This manuscript is a diploma undergraduate thesis written in Greek; Research was conducted at the University of Thrace at the Department of Molecular Biology and Genetics. Hepatoma Up Regulated Protein (HURP) is a microtubule-associated protein essential for the assembly of the mitotic spindle. HURP binds microtubules via its N-terminal domain which is inhibited by HURP's C-terminal tail. The kinase Aurora A activates HURP by phosphorylating its C-terminus which dissociates from the N-terminal microtubule binding domain. This diploma thesis explores how the autoinhibitory interaction between the N- and C-termini of HURP affect binding to proteins with key functions in spindle assembly (e.g. the mitotic motor Eg5 and the microtubule associated proteins TPX2 andXMAP125). I used molecular cloning techniques to generate recombinant GST- and His tagged chimeras of HURP's N- and C-termini. I optimized conditions for the expression of these proteins in bacteria and their purification by liquid chromatography methods using an FPLC system. I performed pull-down assays by incubating the recombinant proteins with HeLa cell extracts and ran western blots to examine the interactions of HURP with Eg5 TPX2 and XMAP125.