Exceptional overproduction of a functional human membrane protein

Maria Nyblom, Fredrik Öberg, Karin Lindkvist-Petersson, Karin Hallgren, Heather Findlay, Jennie Wikström, Anders Karlsson, Örjan Hansson, Paula J Booth, Roslyn M. Bill, Richard Neutze, Kristina Hedfalk*

*Corresponding author for this work

Research output: Contribution to journalArticle

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. 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. © 2007 Elsevier Inc. All rights reserved.

Original languageEnglish
Pages (from-to)110-120
Number of pages11
JournalProtein Expression and Purification
Volume56
Issue number1
DOIs
Publication statusPublished - Nov 2007

Keywords

  • crystallisation
  • fermentation
  • mass spectrometry
  • overproduction
  • pichia pastoris
  • proteoliposomes

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    Nyblom, M., Öberg, F., Lindkvist-Petersson, K., Hallgren, K., Findlay, H., Wikström, J., Karlsson, A., Hansson, Ö., Booth, P. J., Bill, R. M., Neutze, R., & Hedfalk, K. (2007). Exceptional overproduction of a functional human membrane protein. Protein Expression and Purification, 56(1), 110-120. https://doi.org/10.1016/j.pep.2007.07.007