Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Show others and affiliations
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, e0134041Article in journal (Refereed) Published
Abstract [en]

Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in Scerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in Scerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.

Place, publisher, year, edition, pages
2015. Vol. 10, no 7, e0134041
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-119222DOI: 10.1371/journal.pone.0134041ISI: 000358595900071OAI: oai:DiVA.org:su-119222DiVA: diva2:844006
Available from: 2015-08-03 Created: 2015-08-03 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins
Open this publication in new window or tab >>Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biochemical studies of proteins are crucial for a more detailed view of the world around us. The focus of biochemical studies can vary, from a complex mammalian system to a more simple viral entity, but the same methods and principles apply. In biochemistry one rely on both in vitro and in vivo analyses to understand biological processes. Protein crystallography has since the late 1950s been an additional important tool. By visualizing the structures of molecules involved in a biological process one can truly comprehend the molecular mechanisms of an organism or cell at the chemical level. This thesis includes structural biochemical work in combination with mutational and functional studies of proteins from both human and virus.

Human tetraspanins are integral membrane proteins grouped by their conserved structural features. Many of them have been shown to regulate cell migration, fusion, and signalling in the cell by functioning as organizers of multi-molecular membrane complexes. Several tetraspanins are also implicated in different forms of human cancers. How tetraspanins perform their function is still not known at the molecular level and today very little structural data exist on complete tetraspanin proteins. Structural biochemical studies require mg quantities of purified protein, something that is not easily obtained for membrane proteins. This thesis includes a family-wide approach to achieve full-length tetraspanins for biochemical studies. To facilitate this process a GFP-based optimization scheme for production and purification of membrane proteins in E. coli and S. cerevisiae has been applied. By utilizing this approach, we identified 8 human tetraspanins that can be produced and isolated from either E. coli or S. cerevisiae, and in one case using either system.

The temperate bacteriophage P2 is a virus, which can enter both the lytic and the lysogenic cycle upon infection of its host. The outcome of the infection is regulated by and dependent on several proteins encoded by the viral genome. The immunity repressor P2 and the Cox repressor direct the phage into either cycle. Integration and excision of the virus DNA requires the enzyme P2 integrase. The work in this thesis presents high-resolution crystal structures of these key proteins from the regulation of lysogeny in bacteriophage P2. By using a crystallographic approach in combination with mutational studies, key characteristics of these three proteins are presented. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 52 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-119225 (URN)978-91-7649-209-3 (ISBN)
Public defence
2015-09-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2015-08-27 Created: 2015-08-03 Last updated: 2015-08-20Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Skaar, KarinKorza, Henryk J.Högbom, Martin
By organisation
Department of Biochemistry and Biophysics
In the same journal
PLoS ONE
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 49 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf