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 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.
Nyblom
Maria
M
Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-405 30 Göteborg, Sweden.
Oberg
Fredrik
F
Lindkvist-Petersson
Karin
K
Hallgren
Karin
K
Findlay
Heather
H
Wikström
Jennie
J
Karlsson
Anders
A
Hansson
Orjan
O
Booth
Paula J
PJ
Bill
Roslyn M
RM
Neutze
Richard
R
Hedfalk
Kristina
K
D000595:Amino Acid Sequence; D051398:Aquaporin 1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D011994:Recombinant Proteins; D051398:Aquaporin 1