Targeting of Proteins Derived from Self‐Processing Polyproteins Containing Multiple Signal Sequences

Abstract

The 18aa 2A self-cleaving oligopeptide from foot-andmouth 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, selfprocessing 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.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

“…A potential disadvantage in the use of 2A peptides is the residual amino acids left on the C terminus of the first protein with the potential for inappropriate targeting of proteins to different subcellular organelles. 52 The residual 2A peptide left on the a-chain did not appear to significantly affect TCR function in comparison to an a-chain without these residues (Figure 3), but this observation is likely gene specific as we have observed the loss of biological activity of cytokine genes produced using 2A peptides (data not shown).…”

Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver

“…The receptor consists of the murine extracellular and transmembrane domains and the human intracellular domain (Supplementary Figure 1A) and shows fully transforming potential in murine cells. 25 We linked the KIT cDNA to a green fluorescent protein (GFP) coding sequence from copepod Pontellina plumata by a viral 2A-sequence 28 to generate the GFP-2A-KIT D818V fusion protein, hereafter called KIT D816V . During translation the 2A-peptide mediates separation of GFP and KIT, allowing reliable tracking with minimal alteration of the receptor.…”

Engineering Mammalian Mucin-type O-Glycosylation in Plants

“…Other factors may be inefficient 2A sequence cleavage and aberrant localization of the individual proteins. For example, de Felipe and Ryan (52) reported that a two times 2A construct encoding a Golgi-targeted cyan fluorescence protein, Golgitargeted YFP, and a cytosolic puromycin resistance protein (i.e. GT-CFP-2A-GT-YFP-2A-PAC), resulted in Golgi-localized cyan fluorescence protein and YFP aberrantly localized in the mitochondria.…”