Exceptional overproduction of a functional human membrane protein
Artikel i vetenskaplig tidskrift, 2007

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.

NMR STRUCTURE

proteoliposomes

ELECTRON CRYSTALLOGRAPHY

CHIP28 WATER CHANNEL

DRUG DISCOVERY

YEAST PICHIA-PASTORIS

SACCHAROMYCES-CEREVISIAE

Pichia pastoris

STRUCTURAL BASIS

MOLECULAR-BASIS

fermentation

overproduction

crystallisation

CRYSTAL-STRUCTURE

mass spectrometry

ACETYLCHOLINE-RECEPTOR

Författare

Anna Maria Nyblom

Chalmers, Kemi- och bioteknik, Molekylär bioteknik

Fredrik Öberg

Göteborgs universitet

Karin Lindkvist-Petersson

Göteborgs universitet

Karin Hallgren

Göteborgs universitet

H. Findlay

University of Bristol

J. Wikstrom

Chalmers University of Technology

A. Karlsson

Chalmers University of Technology

Örjan Hansson

Göteborgs universitet

P. J. Booth

University of Bristol

R Bill

Aston University

Richard Neutze

Chalmers, Kemi- och bioteknik, Molekylär bioteknik

Göteborgs universitet

Kristina Hedfalk

Göteborgs universitet

Chalmers, Kemi- och bioteknik, Molekylär bioteknik

Protein Expression and Purification

1046-5928 (ISSN) 1096-0279 (eISSN)

Vol. 56 110-120

Ämneskategorier

Kemi

DOI

10.1016/j.pep.2007.07.007

PubMed

17869538