Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant ma...

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Main Authors:	Bergeron, Marc J., Boggavarapu, Rajendra, Meury, Marcel, Ucurum, Zöhre, Caron, Luc, Isenring, Paul, Hediger, Matthias A., Fotiadis, Dimitrios
Format:	Online
Language:	English
Published:	Public Library of Science 2011
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131388/

id	pubmed-3131388
recordtype	oai_dc
spelling	pubmed-31313882011-07-14 Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins Bergeron, Marc J. Boggavarapu, Rajendra Meury, Marcel Ucurum, Zöhre Caron, Luc Isenring, Paul Hediger, Matthias A. Fotiadis, Dimitrios Research Article Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression. Public Library of Science 2011-07-07 /pmc/articles/PMC3131388/ /pubmed/21760919 http://dx.doi.org/10.1371/journal.pone.0021901 Text en Bergeron et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Bergeron, Marc J. Boggavarapu, Rajendra Meury, Marcel Ucurum, Zöhre Caron, Luc Isenring, Paul Hediger, Matthias A. Fotiadis, Dimitrios
spellingShingle	Bergeron, Marc J. Boggavarapu, Rajendra Meury, Marcel Ucurum, Zöhre Caron, Luc Isenring, Paul Hediger, Matthias A. Fotiadis, Dimitrios Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
author_facet	Bergeron, Marc J. Boggavarapu, Rajendra Meury, Marcel Ucurum, Zöhre Caron, Luc Isenring, Paul Hediger, Matthias A. Fotiadis, Dimitrios
author_sort	Bergeron, Marc J.
title	Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
title_short	Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
title_full	Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
title_fullStr	Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
title_full_unstemmed	Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
title_sort	frog oocytes to unveil the structure and supramolecular organization of human transport proteins
description	Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.
publisher	Public Library of Science
publishDate	2011
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131388/
_version_	1611464215808180224

Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

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