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

AUTHOR Ka, Eva
PUBLISHER Grin Verlag (04/19/2016)
PRODUCT TYPE Paperback (Paperback)

Description
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.
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ISBN-13: 9783668190436
ISBN-10: 3668190437
Binding: Paperback or Softback (Trade Paperback (Us))
Content Language: Greek, Modern (after 1453)
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Page Count: 108
Carton Quantity: 64
Product Dimensions: 5.83 x 0.26 x 8.27 inches
Weight: 0.33 pound(s)
Country of Origin: US
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BISAC Categories
Science | Life Sciences - Microbiology
Science | Microbiology
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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.
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