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Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris

Karlsson, M. and Fotiadis, D. and Sjovall, S. and Johansson, I. and Hedfalk, K. and Engel, A. and Kjellbom, P.. (2003) Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris. FEBS letters, Vol. 537, H. 1-3. pp. 68-72.

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Official URL: http://edoc.unibas.ch/dok/A5262395

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Abstract

The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C-terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation-mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix-assisted laser desorption ionization-mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea- and alkali-stripped Pichia membranes by octyl-beta-D-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Engel)
UniBasel Contributors:Engel, Andreas H
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Elsevier Science
ISSN:0014-5793
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:21
Deposited On:22 Mar 2012 13:23

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