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

Dmitry Bratanov; Taras Balandin; Ekaterina Round; Vitaly Shevchenko; Ivan Gushchin; Vitaly Polovinkin; Valentin Borshchevskiy; Valentin Gordeliy

doi:10.1371/journal.pone.0128390

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

PLoS One. 2015 Jun 5;10(6):e0128390. doi: 10.1371/journal.pone.0128390. eCollection 2015.

Authors

Dmitry Bratanov¹, Taras Balandin², Ekaterina Round³, Vitaly Shevchenko⁴, Ivan Gushchin⁵, Vitaly Polovinkin⁵, Valentin Borshchevskiy⁴, Valentin Gordeliy⁵

Affiliations

¹ Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Jülich, Jülich, Germany; Institute of Crystallography, University of Aachen (RWTH), Jägerstrasse 17-19, Aachen, Germany.
² Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Jülich, Jülich, Germany.
³ Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Jülich, Jülich, Germany; Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, IBS, Grenoble, France; CEA, IBS, Grenoble, France.
⁴ Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Jülich, Jülich, Germany; Research-Educational Centre "Bionanophysics", Moscow Institute of Physics and Technology, Dolgoprudniy, Russia.
⁵ Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Jülich, Jülich, Germany; Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, IBS, Grenoble, France; CEA, IBS, Grenoble, France; Research-Educational Centre "Bionanophysics", Moscow Institute of Physics and Technology, Dolgoprudniy, Russia.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Bacteriorhodopsins / genetics
Bacteriorhodopsins / metabolism*
Crystallography, X-Ray
Escherichia coli / metabolism
Halobacterium salinarum / metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Nucleic Acid Conformation
Protein Structure, Tertiary
RNA, Messenger / chemistry
Recombinant Proteins / biosynthesis
Recombinant Proteins / chemistry
Recombinant Proteins / isolation & purification
Sequence Alignment

Substances

RNA, Messenger
Recombinant Proteins
Bacteriorhodopsins

Grants and funding

The work was supported by the program "Chaires d'excellence" edition 2008 of ANR France, CEA(IBS) – HGF(FZJ) STC 5.1 specific agreement; the MC grant (Marie Curie, FP7-PEOPLE-2007-1-1-ITN, project SBMPs); and an EC FP7 grant for the EDICT consortium (HEALTH-201924). Part of this work was supported by BMBF (PhoNa – Photonic Nanomaterials). The work was partially supported by the 5 top100-program of the Ministry for science and education of Russia and by RFBR, research project No. 13-04-91320. The authors greatly acknowledge support of this work by ONEXIM, Russia. The work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.