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Cloning, overexpression and biophysical characterization of grd/grl/wrt domains from Caenorhabditis elegans in Escherichia coli
Södertörn University College, School of Life Sciences.
2008 (English)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.

Place, publisher, year, edition, pages
2008. , p. 25
Keyword [en]
groundhog, warthog, ground-like, disulfide
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:sh:diva-1996OAI: oai:DiVA.org:sh-1996DiVA, id: diva2:139638
Presentation
(English)
Uppsok
fysik/kemi/matematik
Supervisors
Examiners
Available from: 2009-01-26 Created: 2009-01-25 Last updated: 2009-03-02Bibliographically approved

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fulltext(647 kB)272 downloads
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CiteExportLink to record
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Citation style
  • apa
  • ieee
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Output format
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