• Open Access

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

Authors

  • Pablo de Felipe,

    1. Centre for Biomolecular Sciences, North Haugh, University of St. Andrews, St. Andrews, Scotland, UK
    2. Spanish Medicines Agency (AEMPS), Parque Empresarial “Las Mercedes”, Campezo 1 – Edificio 8, Madrid, Spain
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  • Garry A. Luke,

    1. Centre for Biomolecular Sciences, North Haugh, University of St. Andrews, St. Andrews, Scotland, UK
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  • Jeremy D. Brown,

    1. RNA Biology Group and Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, UK
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  • Martin D. Ryan Professor

    Corresponding author
    1. Centre for Biomolecular Sciences, North Haugh, University of St. Andrews, St. Andrews, Scotland, UK
    • Centre for Biomolecular Sciences, North Haugh, University of St. Andrews, KY16 9ST, St. Andrews, Scotland, UK, Fax: +44-1334-462595
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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 ‘slipstream’ 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.

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