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A simple strategy towards membrane protein purification and crystallization
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Pär Nordlund)
2006 (English)In: International Journal of Biological Macromolecules, ISSN 0141-8130, Vol. 39, no 1-3, 83-87 p.Article in journal (Refereed) Published
Abstract [en]

A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.

Place, publisher, year, edition, pages
2006. Vol. 39, no 1-3, 83-87 p.
Keyword [en]
membrane proteins, detergent screen, high-throughput
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Structural Biology
Identifiers
URN: urn:nbn:se:su:diva-29986DOI: 10.1016/j.ijbiomac.2006.02.011OAI: oai:DiVA.org:su-29986DiVA: diva2:241099
Available from: 2009-10-01 Created: 2009-09-27 Last updated: 2009-10-01Bibliographically approved
In thesis
1. Structural biology of integral membrane proteins - From methods to molecular mechanisms
Open this publication in new window or tab >>Structural biology of integral membrane proteins - From methods to molecular mechanisms
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure.  Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis  are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials.

Despite the challenges in membrane protein structure determination two structures are presented in the thesis:

The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway.

The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy, 2009. 59 p.
Keyword
membrane proteins, CorA, magnesium transport, screening, Leukotriene C4 synthase, detergents
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-30069 (URN)978-91-628-7899-3 (ISBN)
Public defence
2009-10-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2009-10-07 Created: 2009-10-01 Last updated: 2012-08-10Bibliographically approved

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