Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris.
Journal article, 2003
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.
ultrastructure
genetics
physiology
Oocytes
Kinetics
Spinacia oleracea
DNA Primers
isolation & purification
physiology
metabolism
Animals
genetics
physiology
Serine
Spectrometry
ultrastructure
physiology
Matrix-Assisted Laser Desorption-Ionization
metabolism
genetics
Plant Proteins
Aquaporins
Mass
physiology
Xenopus laevis
Proteolipids
ultrastructure
Recombinant Proteins
Phosphoserine
ultrastructure
Ion Channels
genetics
Base Sequence
Pichia
Phosphorylation
Female
Author
Maria Karlsson
Dimitrios Fotiadis
Sara Sjövall
Ingela Johansson
Kristina Hedfalk
Chalmers, Department of Chemistry and Bioscience, Molecular Biotechnology
Andreas Engel
Per Kjellbom
FEBS Letters
0014-5793 (ISSN) 18733468 (eISSN)
Vol. 537 1-3 68-72Subject Categories
Biological Sciences
PubMed
12606033