Send to

Choose Destination
Biotechnol J. 2010 Feb;5(2):213-23. doi: 10.1002/biot.200900134.

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

Author information

Centre for Biomolecular Sciences, North Haugh, University of St. Andrews, St. Andrews, Scotland, UK.


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.

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Wiley Icon for PubMed Central
Loading ...
Support Center