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Cell-free productio...
Cell-free production and characterisation of human uncoupling protein 1–3
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- Rebuffet, Etienne (författare)
- Gothenburg University,Göteborgs universitet,University of Gothenburg,Institutionen för kemi och molekylärbiologi,Department of Chemistry and Molecular Biology
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- Frick, Anna, 1982 (författare)
- Gothenburg University,Göteborgs universitet,University of Gothenburg,Institutionen för kemi och molekylärbiologi,Department of Chemistry and Molecular Biology
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- Järvå, Michael (författare)
- Gothenburg University,Göteborgs universitet,University of Gothenburg,Institutionen för kemi och molekylärbiologi,Department of Chemistry and Molecular Biology
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- Törnroth-Horsefield, Susanna (författare)
- Gothenburg University,Göteborgs universitet,University of Gothenburg,Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH,Institutionen för kemi och molekylärbiologi,Department of Chemistry and Molecular Biology
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(creator_code:org_t)
- Elsevier BV, 2017
- 2017
- Engelska 6 s.
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Ingår i: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 10, s. 276-281
- Relaterad länk:
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http://dx.doi.org/10... (free)
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https://doi.org/10.1...
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https://lup.lub.lu.s...
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https://doi.org/10.1...
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https://gup.ub.gu.se...
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Abstract
Ämnesord
Stäng
- The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer. Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Biofysik (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biophysics (hsv//eng)
Nyckelord
- Cell-free expression
- Membrane protein
- Mitochondrial carrier
- Uncoupling protein
Publikations- och innehållstyp
- art (ämneskategori)
- ref (ämneskategori)
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