Inhibition of 2A‐mediated ‘cleavage’ of certain artificial polyproteins bearing N‐terminal signal sequences

Abstract

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.

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

“…There are four widely used 2A sequences, which are derived from the foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), Thoseaasigna virus (T2A), and equine rhinitis A virus (E2A), with P2A as the most effective one [98]. 2A is active in all eukaryotic cells [99], but not in prokaryotes [100]. 2A sequences (54-66 bp) are short compared to IRES and internal promoters, and therefore provide an advantage in vector design because of the limited packaging capacity of the widely used lentiviral vectors and RVs.…”

Translating 2A Research into Practice

“…The residues that influence cleavage are predicted to reside within the translocon; this length may allow interactions between the nascent peptide and the ribosome that lead to inhibition of the 2A reaction (Ménétret et al, 2000;Beckmann et al, 2001;de Felipe et al, 2010). Solutions to the problem include the use of longer versions of 2A with extra sequences derived from the capsid protein ("1D") (Ryan et al, 1991;Groot Bramel-Verheije et al, 2000;Donnelly et al, 2001b;Klump et al, 2001).…”

“…First, antibodies to the 2A-peptide have been generated, allowing detection and/or immunoprecipitation of "upstream" protein products derived from 2A-containing transgenes . Second, a shift in protein size is observed in 2A-tagged proteins which can be useful if mutant and endogenous proteins are co-expressed and need to be identified (Szmczak et al, 2004;de Felipe et al, 2010). To our knowledge, the presence of a proline attached to the amino-terminus of the second protein, as a relic of the 2A self-cleaving process, does not interfere significantly with activity and trafficking; it does, however, confer high protein stability (Varshavsky, 1992).…”

“…Another approach is to detect the expressed protein through its activity (conversion of a substrate in a fluorescent product as with β-galactosidase). We (Halpin et al, 1999;Funston et al, 2008;de Felipe et al, 2010) and others (Samalova et al, 2006(Samalova et al, & 2008Hasegawa et al, 2007;Torres et al, 2010) have successfully used the 2A sequence in a number of in vitro and in vivo heterologous systems to achieve production of various combinations of fluorescent proteins and proteins requiring discrete co-and post-translational subcellular localization. The zebrafish (Danio rerio) has proved to be an excellent vertebrate model system for basic and biomedical science and comparative genomics.…”

A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact

“…In addition, tagging of viral proteins, either at the C or N terminus, is likely to be detrimental for virus replication (12,16). To overcome this issue, I used the cis-acting hydrolase element (CHYSEL) (20)(21)(22)(23). This strategy is based on the insertion of the 2A ribosomal skipping site, which provides an efficient way to express comparable levels and physically separate proteins from a single promoter.…”