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GFP as a tool to monitor membrane protein topology and overexpression in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2005. , 65 p.
Keyword [en]
GFP, membrane protein, topology, overexpression, high-throughput
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-734ISBN: 91-7155-160-3 (print)OAI: oai:DiVA.org:su-734DiVA: diva2:198074
Public defence
2005-12-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2005-11-09 Created: 2005-11-09 Last updated: 2012-02-06Bibliographically approved
List of papers
1. Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis
Open this publication in new window or tab >>Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis
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2002 In: Proc. Natl. Acad. Sci., Vol. 99, no 5, 2690-2695 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24666 (URN)
Note
Part of urn:nbn:se:su:diva-734Available from: 2005-11-09 Created: 2005-11-09Bibliographically approved
2. Experimentally based topology models for E. coli inner membrane proteins
Open this publication in new window or tab >>Experimentally based topology models for E. coli inner membrane proteins
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2004 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 4, 937-945 p.Article in journal (Refereed) Published
Abstract [en]

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.

Keyword
membrane proteins, topology, prediction, bioinformatics, fusion protein, PhoA, green fluorescent, protein, FP
Identifiers
urn:nbn:se:su:diva-24327 (URN)10.1110/ps.03553804 (DOI)
Note
Part of urn:nbn:se:su:diva-6875Available from: 2007-05-24 Created: 2007-05-15 Last updated: 2017-12-13Bibliographically approved
3. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli
Open this publication in new window or tab >>Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli
2001 In: FEBS Lett., ISSN 0014-5793, Vol. 507, no 2, 220-224 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24668 (URN)
Note
Part of urn:nbn:se:su:diva-734Available from: 2005-11-09 Created: 2005-11-09Bibliographically approved
4. A scalable, GFP-based pipeline for membrane protein overexpression screening and purification
Open this publication in new window or tab >>A scalable, GFP-based pipeline for membrane protein overexpression screening and purification
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2005 In: Protein Sci., Vol. 14, no 8, 2011-2017 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24669 (URN)
Note
Part of urn:nbn:se:su:diva-734Available from: 2005-11-09 Created: 2005-11-09Bibliographically approved

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