Exceptional overproduction of a functional human membrane protein

• 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.

Subject headings

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Keyword

overproduction

Pichia pastoris

mass spectrometry

crystallisation

proteoliposomes

fermentation

YEAST PICHIA-PASTORIS

CHIP28 WATER CHANNEL

CRYSTAL-STRUCTURE

STRUCTURAL BASIS

ELECTRON CRYSTALLOGRAPHY

SACCHAROMYCES-CEREVISIAE

ACETYLCHOLINE-RECEPTOR

MOLECULAR-BASIS

DRUG DISCOVERY

NMR STRUCTURE

DRUG DISCOVERY

Publication and Content Type

ref (subject category)

art (subject category)