Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences.
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.
de Felipe
Pablo
P
Centre for Biomolecular Sciences, North Haugh, University of St. Andrews, St. Andrews, Scotland, UK.
Luke
Garry A
GA
Brown
Jeremy D
JD
Ryan
Martin D
MD
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D020815:Polyproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046188:Protein Modification, Translational; D021382:Protein Sorting Signals; D021381:Protein Transport; D017384:Sequence Deletion
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D020815:Polyproteins; D021382:Protein Sorting Signals; C054562:yellow fluorescent protein, Bacteria; D003546:Cysteine Endopeptidases