Overcoming the toxicity of membrane peptide expression in bacteria by upstream insertion of Asp-Pro sequence

Biochim Biophys Acta. 2004 Jan 28;1660(1-2):53-65. doi: 10.1016/j.bbamem.2003.10.013.

Abstract

Transmembrane (TM) peptides often induce toxic effects when expressed in bacteria, probably due to membrane destabilization. We report here that in the case of the TM domains of hepatitis C virus (HCV) E1 and E2 envelope proteins, which are both particularly toxic for the bacteria, the insertion of the Asp-Pro (DP) sequence dramatically reduced their toxicities and promoted their expressions when produced as glutathione S-transferase (GST) GST-DP-TM chimeras. Subcellular fractionation showed that these chimeras co-sediment with the membrane fraction and contain active GST that could be solubilized with a mild detergent. Surprisingly, immuno-gold electron microscopy clearly showed that such chimeras are not localized in the membrane but in the cytosol. We thus postulate that they likely form proteo-lipidic aggregates, which prevent the bacteria from toxicity by sequestering the TM part of the chimeras. The reduction of toxicity in the presence of the Asp-Pro sequence is possibly due to Asp's negative charge that probably disadvantages the binding of the TM peptides to the membrane. In addition, the structural features of Pro residue could promote the formation of chimera aggregates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid / genetics
  • Cell Fractionation
  • Cloning, Molecular
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism*
  • Escherichia coli / ultrastructure
  • Glutathione Transferase / chemistry
  • Membrane Proteins / biosynthesis*
  • Microscopy, Immunoelectron
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Peptides / chemistry
  • Peptides / genetics
  • Proline / genetics
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Serine Endopeptidases
  • Viral Envelope Proteins / biosynthesis

Substances

  • E1 protein, Hepatitis C virus
  • Membrane Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Viral Envelope Proteins
  • glycoprotein E2, Hepatitis C virus
  • Aspartic Acid
  • Proline
  • Glutathione Transferase
  • Serine Endopeptidases
  • type I signal peptidase