Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences.
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.
de Felipe
Pablo
P
School of Biology, Centre for Biomolecular Sciences, Biomolecular Sciences Building, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK.
Ryan
Martin D
MD
D000818:Animals; D006367:HeLa Cells; D006801:Humans; D016366:Open Reading Frames; D046188:Protein Modification, Translational; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C057421:virus protein 2A