Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris.
Artikel i vetenskaplig tidskrift, 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

Författare

Dimitrios Fotiadis

Sara Sjövall

Ingela Johansson

Kristina Hedfalk

Chalmers, Institutionen för kemi och biovetenskap, Molekylär bioteknik

Andreas Engel

Per Kjellbom

FEBS Letters

0014-5793 (ISSN)

Vol. 537 1-3 68-72

Ämneskategorier

Biologiska vetenskaper

PubMed

12606033