Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences

Pablo De Felipe, Garry Alec Luke, Jeremy D. Brown, Martin Denis Ryan

Research output: Contribution to journalArticlepeer-review

57 Citations (Scopus)

Abstract

Where 2A oligopeptide sequences occur within ORFs, the formation of the glycyl-prolyl peptide bond at the C-terminus of (each) 2A does not occur. This property can be used to concatenate sequences encoding several proteins into a single ORF: each component of such an artificial polyprotein is generated as a discrete translation product. 2A and '2A-like' sequences have become widely utilised in biotechnology and biomedicine. Individual proteins may also be co- and post-translationally targeted to a variety of sub-cellular sites. In the case of polyproteins bearing N-terminal signal sequences we observed, however, that the protein downstream of 2A (no signal) was translocated into the endoplasmic reticulum (ER). We interpreted these data as a form of 'slip-stream' translocation: downstream proteins, without signals, were translocated through a translocon pore already formed by the signal sequence at the N-terminus of the polyprotein. Here we show this effect is, in fact, due to inhibition of the 2A reaction (formation of fusion protein) by the C-terminal region (immediately upstream of 2A) of some proteins when translocated into the ER. Solutions to this problem include the use of longer 2As (with a favourable upstream context) or modifying the order of proteins comprising polyproteins.

Original languageEnglish
Pages (from-to)213-223
Number of pages11
JournalBiotechnology Journal
Volume5
Issue number2
DOIs
Publication statusPublished - Feb 2010

Keywords

  • 2A
  • Polyprotein
  • Skipping
  • Slipstream
  • Targeting
  • MOUTH-DISEASE VIRUS
  • EMBRYONIC STEM-CELLS
  • RIBOSOME-CHANNEL COMPLEX
  • PREPRO-ALPHA-FACTOR
  • OPEN READING FRAME
  • HUMAN T-CELLS
  • ENDOPLASMIC-RETICULUM
  • PROTEIN TRANSLOCATION
  • THERAPEUTIC LEVELS
  • 2A-LIKE SEQUENCES

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