Advanced

Exceptional overproduction of a functional human membrane protein

Nyblom, Maria; Öberg, Fredrik; Lindkvist-Petersson, Karin LU ; Hallgren, Karin; Findlay, Heather; Wikström, Jennie; Karlsson, Anders; Hansson, Örjan; Booth, Paula J. and Bill, Roslyn M, et al. (2007) In Protein Expression and Purification 56(1). p.110-120
Abstract

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.... (More)

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 hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 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.

(Less)
Please use this url to cite or link to this publication:
author
, et al. (More)
(Less)
publishing date
type
Contribution to journal
publication status
published
keywords
Crystallisation, Fermentation, Mass spectrometry, Overproduction, Pichia pastoris, Proteoliposomes
in
Protein Expression and Purification
volume
56
issue
1
pages
11 pages
publisher
Academic Press
external identifiers
  • scopus:34848820557
ISSN
1046-5928
DOI
10.1016/j.pep.2007.07.007
language
English
LU publication?
no
id
1e95845c-8ded-401a-af4f-034e0e1c81e3
date added to LUP
2017-02-15 15:23:45
date last changed
2017-05-28 04:57:34
@article{1e95845c-8ded-401a-af4f-034e0e1c81e3,
  abstract     = {<p>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 hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 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.</p>},
  author       = {Nyblom, Maria and Öberg, Fredrik and Lindkvist-Petersson, Karin and Hallgren, Karin and Findlay, Heather and Wikström, Jennie and Karlsson, Anders and Hansson, Örjan and Booth, Paula J. and Bill, Roslyn M and Neutze, Richard and Hedfalk, Kristina},
  issn         = {1046-5928},
  keyword      = {Crystallisation,Fermentation,Mass spectrometry,Overproduction,Pichia pastoris,Proteoliposomes},
  language     = {eng},
  number       = {1},
  pages        = {110--120},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Exceptional overproduction of a functional human membrane protein},
  url          = {http://dx.doi.org/10.1016/j.pep.2007.07.007},
  volume       = {56},
  year         = {2007},
}