Overcoming the toxicity of membrane peptide expression in bacteria by upstream insertion of Asp-Pro sequence.
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.
Montigny
Cédric
C
Centre d'études de Saclay, Département de Biologie Joliot Curie, Section de Biophysique des Fonctions Membranaires, Unité de Recherche Associée 2096 of the Centre National de la Recherche Scientifique, Gif-sur-Yvette cedex, France.
Penin
François
F
Lethias
Claire
C
Falson
Pierre
P
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D002458:Cell Fractionation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D005982:Glutathione Transferase / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases; D014759:Viral Envelope Proteins / Q000096:biosynthesis
C087035:E1 protein, Hepatitis C virus; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D001224:Aspartic Acid; D011392:Proline; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; C026515:type I signal peptidase