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Targeting of Proteins Derived from Self‐Processing Polyproteins Containing Multiple Signal Sequences
Author(s) -
de Felipe Pablo,
Ryan Martin D.
Publication year - 2004
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1398-9219.2004.00205.x
Subject(s) - polyproteins , signal peptide , biology , endoplasmic reticulum , microbiology and biotechnology , green fluorescent protein , protein sorting signals , translocon , signal peptidase , internal ribosome entry site , ribosome , peptide sequence , biochemistry , rna , membrane protein , membrane , gene
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.