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A rapid expression and purification condition screening protocol for membrane protein structural biology
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-2994-5839
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Number of Authors: 52017 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 26, no 8, p. 1653-1666Article in journal (Refereed) Published
Abstract [en]

Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.

Place, publisher, year, edition, pages
2017. Vol. 26, no 8, p. 1653-1666
Keywords [en]
membrane protein, E. coli, FSEC, GFP, detergent screening, IMAC purification, structural biology
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-145854DOI: 10.1002/pro.3196ISI: 000406123600018OAI: oai:DiVA.org:su-145854DiVA, id: diva2:1135607
Available from: 2017-08-23 Created: 2017-08-23 Last updated: 2019-12-09Bibliographically approved
In thesis
1. Structural and Functional Studies of Membrane Proteins: From Characterisation of a Fatty Acyl-CoA Synthetase to the Discovery of Superoxide Oxidase
Open this publication in new window or tab >>Structural and Functional Studies of Membrane Proteins: From Characterisation of a Fatty Acyl-CoA Synthetase to the Discovery of Superoxide Oxidase
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is divided into three parts; the first part describes a method for efficient screening of membrane proteins for crystallography. By utilising the properties of a folding reporter GFP it is possible to quickly and accurately screen the stability of a protein in a range of conditions without full purification. This allows rapid assessment of the suitability of a protein for crystallography and a parallel optimisation of purification conditions for subsequent large-scale protein production.

The second part describes the discovery of a membrane bound superoxide oxidase (SOO), a novel scavenger of membrane proximal superoxide. SOO is a kinetically perfect enzyme, reacting at rates close to the diffusion limit in a similar fashion to other superoxide scavengers, such as superoxide dismutase. We propose that SOO rescues electrons “lost” to superoxide and recycles them back into the respiratory chain, releasing oxygen. At the same time SOO contributes to the proton motive force by uptake of protons from the cytoplasmic side of the membrane.

The third part concerns the fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis (M. tuberculosis). It represents a critical node point in M. tuberculosis lipid metabolism and has been suggested to be a vital component of M. tuberculosis survival in host cell macrophages. FadD13 harbours a hydrophobic cavity that is unable to house the very-long-chain substrates the enzyme has preference for. We propose that FadD13 is a peripheral membrane protein, utilising the membrane to house the very-long-chain fatty acid substrates during the activation reaction.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 74
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167812 (URN)978-91-7797-648-6 (ISBN)978-91-7797-649-3 (ISBN)
Public defence
2019-05-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2019-05-06 Created: 2019-04-04 Last updated: 2019-05-02Bibliographically approved

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