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Structural description of a hexa-His tag chelated with two metal ions - molecular basis of protein poly-His tag IMAC chromatography 
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Strukturbiokemi)
(English)Manuscript (Other academic)
Abstract [en]

In this work we describe the molecular basis for binding of poly histidine tagged protein to IMAC resins such as IDA or NTA and explain early experimental findings on the affinity of various His tags. 

National Category
Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-27062OAI: oai:DiVA.org:su-27062DiVA: diva2:212548
Note
Totalt antal författare: 6 Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2010-01-14Bibliographically 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

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