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Novel Technologies for Recombinant Protein Overexpression in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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: urn:nbn:se:su:diva-861ISBN: 91-7155-212-X (print)OAI: oai:DiVA.org:su-861DiVA: diva2:200549
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
List of papers
1. The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH
Open this publication in new window or tab >>The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH
2005 In: Biochimica et Biophysica Acta, Vol. 1707, no 2-3, 179-88 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25799 (URN)
Note
Part of urn:nbn:se:su:diva-861Available from: 2006-03-03 Created: 2006-03-03Bibliographically approved
2. Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli
Open this publication in new window or tab >>Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli
Show others...
2005 (English)In: Nature Methods, ISSN 1548-7091, Vol. 2, no 7, 507-509 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25545 (URN)
Note
Part of urn:nbn:se:su:diva-8261Available from: 2008-10-23 Created: 2008-10-14 Last updated: 2010-01-13Bibliographically approved
3. An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries
Open this publication in new window or tab >>An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries
Show others...
2006 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, Vol. 65, no 2, 266-273 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25544 (URN)
Note
Part of urn:nbn:se:su:diva-8261Available from: 2008-10-23 Created: 2008-10-14 Last updated: 2010-01-13Bibliographically approved
4. Engineering membrane protein overproduction in Escherichia coli
Open this publication in new window or tab >>Engineering membrane protein overproduction in Escherichia coli
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.

 

National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-27055 (URN)10.1110/ps.073242508 (DOI)000254197800009 ()
Note
Totalt antal författare: 6Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2017-12-13Bibliographically approved

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