Bicistronic mRNAs to enhance membrane protein overexpression

J Mol Biol. 2015 Feb 27;427(4):943-954. doi: 10.1016/j.jmb.2014.11.002. Epub 2014 Nov 9.


Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy, we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.

Keywords: mRNA secondary structure; protein folding; structural biology; transcriptional fusion; ybeL.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / biosynthesis*
  • ATP-Binding Cassette Transporters / genetics
  • Enterococcus faecalis / genetics
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial / genetics*
  • Green Fluorescent Proteins / genetics
  • Membrane Proteins / biosynthesis*
  • Membrane Proteins / genetics
  • Protein Biosynthesis
  • Protein Folding
  • RNA, Messenger / genetics
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Transcription, Genetic / genetics


  • ATP-Binding Cassette Transporters
  • Membrane Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins