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An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allo...

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Autores principales: Bratanov, Dmitry, Balandin, Taras, Round, Ekaterina, Shevchenko, Vitaly, Gushchin, Ivan, Polovinkin, Vitaly, Borshchevskiy, Valentin, Gordeliy, Valentin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457421/
https://www.ncbi.nlm.nih.gov/pubmed/26046789
http://dx.doi.org/10.1371/journal.pone.0128390
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author Bratanov, Dmitry
Balandin, Taras
Round, Ekaterina
Shevchenko, Vitaly
Gushchin, Ivan
Polovinkin, Vitaly
Borshchevskiy, Valentin
Gordeliy, Valentin
author_facet Bratanov, Dmitry
Balandin, Taras
Round, Ekaterina
Shevchenko, Vitaly
Gushchin, Ivan
Polovinkin, Vitaly
Borshchevskiy, Valentin
Gordeliy, Valentin
author_sort Bratanov, Dmitry
collection PubMed
description Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.
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spelling pubmed-44574212015-06-09 An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin Bratanov, Dmitry Balandin, Taras Round, Ekaterina Shevchenko, Vitaly Gushchin, Ivan Polovinkin, Vitaly Borshchevskiy, Valentin Gordeliy, Valentin PLoS One Research Article Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. Public Library of Science 2015-06-05 /pmc/articles/PMC4457421/ /pubmed/26046789 http://dx.doi.org/10.1371/journal.pone.0128390 Text en © 2015 Bratanov 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.
spellingShingle Research Article
Bratanov, Dmitry
Balandin, Taras
Round, Ekaterina
Shevchenko, Vitaly
Gushchin, Ivan
Polovinkin, Vitaly
Borshchevskiy, Valentin
Gordeliy, Valentin
An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin
title An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin
title_full An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin
title_fullStr An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin
title_full_unstemmed An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin
title_short An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin
title_sort approach to heterologous expression of membrane proteins. the case of bacteriorhodopsin
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457421/
https://www.ncbi.nlm.nih.gov/pubmed/26046789
http://dx.doi.org/10.1371/journal.pone.0128390
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