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.

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

Grant support

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.