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Exceptional overpro...
Exceptional overproduction of a functional human membrane protein
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- Nyblom, Anna Maria, 1975 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Öberg, Fredrik, 1982 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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- Lindkvist-Petersson, Karin (author)
- Gothenburg University,Göteborgs universitet,Institutionen för cell- och molekylärbiologi,Department of Cell and Molecular Biology,University of Gothenburg
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- Hallgren, Karin (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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- Findlay, H. (author)
- University of Bristol
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- Wikstrom, J. (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Karlsson, A. (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Hansson, Örjan, 1955 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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- Booth, P. J. (author)
- University of Bristol
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- Bill, Roslyn M. (author)
- Aston University
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- Neutze, Richard, 1969 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg,Chalmers tekniska högskola,Chalmers University of Technology
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- Hedfalk, Kristina, 1969 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,Chalmers tekniska högskola,Chalmers University of Technology,University of Gothenburg
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(creator_code:org_t)
- Elsevier BV, 2007
- 2007
- English.
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 56:1, s. 110-120
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Abstract
Subject headings
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- Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQPI in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. (c) 2007 Elsevier Inc. All rights reserved.
Subject headings
- NATURVETENSKAP -- Kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences (hsv//eng)
Keyword
- overproduction
- Pichia pastoris
- mass spectrometry
- crystallisation
- proteoliposomes
- fermentation
- YEAST PICHIA-PASTORIS
- CHIP28 WATER CHANNEL
- CRYSTAL-STRUCTURE
- STRUCTURAL BASIS
- ELECTRON CRYSTALLOGRAPHY
- SACCHAROMYCES-CEREVISIAE
- ACETYLCHOLINE-RECEPTOR
- MOLECULAR-BASIS
- DRUG DISCOVERY
- NMR STRUCTURE
- DRUG DISCOVERY
Publication and Content Type
- ref (subject category)
- art (subject category)
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- By the author/editor
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Nyblom, Anna Mar ...
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Öberg, Fredrik, ...
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Lindkvist-Peters ...
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Hallgren, Karin
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Findlay, H.
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Wikstrom, J.
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show more...
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Karlsson, A.
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Hansson, Örjan, ...
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Booth, P. J.
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Bill, Roslyn M.
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Neutze, Richard, ...
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Hedfalk, Kristin ...
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- About the subject
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Chemical Science ...
- Articles in the publication
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Protein Expressi ...
- By the university
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University of Gothenburg
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Chalmers University of Technology