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A simple strategy towards membrane protein purification and crystallization
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. (Pär Nordlund)
2006 (engelsk)Inngår i: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 39, nr 1-3, s. 83-87Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2006. Vol. 39, nr 1-3, s. 83-87
Emneord [en]
membrane proteins, detergent screen, high-throughput
HSV kategori
Forskningsprogram
strukturbiologi
Identifikatorer
URN: urn:nbn:se:su:diva-29986DOI: 10.1016/j.ijbiomac.2006.02.011OAI: oai:DiVA.org:su-29986DiVA, id: diva2:241099
Tilgjengelig fra: 2009-10-01 Laget: 2009-09-27 Sist oppdatert: 2017-12-13bibliografisk kontrollert
Inngår i avhandling
1. Structural biology of integral membrane proteins - From methods to molecular mechanisms
Åpne denne publikasjonen i ny fane eller vindu >>Structural biology of integral membrane proteins - From methods to molecular mechanisms
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy, 2009. s. 59
Emneord
membrane proteins, CorA, magnesium transport, screening, Leukotriene C4 synthase, detergents
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-30069 (URN)978-91-628-7899-3 (ISBN)
Disputas
2009-10-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-10-07 Laget: 2009-10-01 Sist oppdatert: 2012-08-10bibliografisk kontrollert

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