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

Karlsson, Maria LU ; Fotiadis, D; Sjövall Larsen, Sara LU ; Johansson, Ingela; Hedfalk, K; Engel, A and Kjellbom, Per LU (2003) In FEBS Letters 537(1-3). p.68-72
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... (More)
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-small beta, Greek-Image-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Aquaporin, Overexpression, Electron microscopy, Mass spectrometry, Reconstitution, Pichia pastoris
in
FEBS Letters
volume
537
issue
1-3
pages
68 - 72
publisher
Wiley-Blackwell
external identifiers
  • pmid:12606033
  • wos:000181304100013
  • scopus:0037468462
ISSN
1873-3468
DOI
10.1016/S0014-5793(03)00082-6
language
English
LU publication?
yes
id
b6c85c06-b895-43d2-8abf-e91d55ead265 (old id 132681)
date added to LUP
2007-07-05 16:53:56
date last changed
2018-08-05 04:03:54
@article{b6c85c06-b895-43d2-8abf-e91d55ead265,
  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-small beta, Greek-Image-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.},
  author       = {Karlsson, Maria and Fotiadis, D and Sjövall Larsen, Sara and Johansson, Ingela and Hedfalk, K and Engel, A and Kjellbom, Per},
  issn         = {1873-3468},
  keyword      = {Aquaporin,Overexpression,Electron microscopy,Mass spectrometry,Reconstitution,Pichia pastoris},
  language     = {eng},
  number       = {1-3},
  pages        = {68--72},
  publisher    = {Wiley-Blackwell},
  series       = {FEBS Letters},
  title        = {Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris.},
  url          = {http://dx.doi.org/10.1016/S0014-5793(03)00082-6},
  volume       = {537},
  year         = {2003},
}