Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences

P deFelipe, Martin Denis Ryan

Research output: Contribution to journalArticlepeer-review

83 Citations (Scopus)

Abstract

The 18aa 2A self-cleaving oligopeptide from foot-and-mouth disease virus can be used for co-expression of multiple, discrete proteins from a single ORF. 2A mediates a co-translational cleavage at its own C-terminus and is proposed to manipulate the ribosome into skipping the synthesis of a specific peptide bond (producing a discontinuity in the peptide backbone), rather than being involved in proteolysis. To explore the utility of the system to target discrete processing products, self-processing polyproteins comprising fluorescent proteins flanking 2A were constructed, permutating both the type of signal sequence and the location within the polyprotein. A polyprotein comprising a protein bearing an N-terminal signal sequence, 2A, then a protein lacking any signal sequence, was constructed. Interestingly, both proteins were translocated into the endoplasmic reticulum. Despite the discontinuity in the peptide backbone, the mammalian ribosome:translocon complex did not disassemble - the second protein (lacking any signal) 'slipstreamed' through the translocon formed by the first (signal-bearing) protein. These polyprotein systems provide a novel method of targeting proteins to different subcellular sites by transfection with a plasmid encoding a single ORF. The inclusion of a fluorescent reporter enables visualisation of expression levels, whilst inclusion of a selectable marker enables stable cell-lines to be established rapidly.

Original languageEnglish
Pages (from-to)616-626
Number of pages11
JournalTraffic
Volume5
Issue number8
DOIs
Publication statusPublished - Aug 2004

Keywords

  • 2A
  • co-translational
  • localisation
  • polyprotein/fluorescent
  • signal
  • targeting
  • ENDOPLASMIC-RETICULUM MEMBRANE
  • RECOGNITION PARTICLE
  • NASCENT POLYPEPTIDE
  • NUCLEAR-LOCALIZATION
  • CLEAVAGE ACTIVITIES
  • CONDUCTING CHANNEL
  • LIVING CELLS
  • ER MEMBRANE
  • TRANSLOCATION
  • VIRUS

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