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Engineering membrane protein overproduction in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Strukturbiokemi)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Strukturbiokemi)
2008 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 17, no 4, 673-680 p.Article in journal (Refereed) Published
Abstract [en]

Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold.

 

Place, publisher, year, edition, pages
2008. Vol. 17, no 4, 673-680 p.
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-27055DOI: 10.1110/ps.073242508ISI: 000254197800009OAI: oai:DiVA.org:su-27055DiVA: diva2:212509
Note
Totalt antal författare: 6Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Membrane Protein Tailoring and Structural Studies of Leukotriene C4 Synthase
Open this publication in new window or tab >>Membrane Protein Tailoring and Structural Studies of Leukotriene C4 Synthase
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Despite a dramatic increase in the number of proteins that have been structurally characterized in recent years, there are still less than 200 unique structures of membrane proteins known today. This is only 1% of the total number of unique protein structures found in structural databases worldwide. There are several reasons for this hindered progress in the structure determination of membrane proteins; it is difficult to generate membrane proteins in recombinant expression systems and it requires the use of appropriate detergents, both for membrane extraction and to keep them stable in solution. This makes isolation and purification problematic. Importantly, once isolated, these proteins are notoriously difficult to crystallize for X-ray structure determination.

 In this thesis, I present two techniques that can be used to increase the likelihood of success in the structural determination of membrane proteins. I started by focusing on problems that occur at an early stage of the process, where I developed a directed-evolution method to overcome problems with low yields during membrane protein production. In addition, I describe a screen for optimal detergent usage when purifying and crystallizing recombinant membrane proteins in eukaryotic hosts.

 The crystal structure of human Leukotriene C4 synthase has been solved. This is the first human membrane protein whose structure has been solved at high resolution. The model provides a structural basis for the formation of potent lipid mediators, which are implicated in the pathophysiology of asthma and chronic inflammation. Furthermore, the structure reveals insight into how specificity can be achieved for lipophilic substrate molecules. In addition, I have determined the crystal structure of a hexahistidine tag and use it to describe the molecular basis of the single most used chromatography method, IMAC.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 68 p.
Keyword
membrane proteins, random mutagenesis, detergent screening, Leukotriene C₄ synthase, IMAC
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-27067 (URN)978-91-7155-882-4 (ISBN)
Public defence
2009-05-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A , Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2009-05-06 Created: 2009-04-22 Last updated: 2009-04-24Bibliographically approved
2. Novel Technologies for Recombinant Protein Overexpression in Escherichia coli
Open this publication in new window or tab >>Novel Technologies for Recombinant Protein Overexpression in Escherichia coli
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of recombinant protein is a cornerstone in many structural and functional studies. The enteric bacterium Escherichia Coli is the most commonly used organism for producing recombinant proteins. E. coli has several advantages over other expression hosts, but also one major disadvantage - the protein of interest does not always adopt its native conformation. Instead the protein might form large insoluble aggregates, inclusion bodies, within the cell. In particular, the heterologous overexpression of eukaryotic and membrane proteins are troublesome.

In this thesis, methods are described that can be used to increase the likelihood of overexpressing eukaryotic proteins as well as membrane proteins. In particular, a novel method is described that can distinguish between bacterial colonies expressing soluble proteins from those expressing inclusion bodies. The method utilizes the fact that inclusion bodies are of a considerable size and can be removed by filtration. Using this screening method in combination with methods that alter the physical properties of proteins, we have shown that the likelihood of overexpression in E. coli can be dramatically increased.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2006. 76 p.
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-861 (URN)91-7155-212-X (ISBN)
Public defence
2006-03-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2006-03-03 Created: 2006-03-03 Last updated: 2010-01-13Bibliographically approved

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