The protein coexpression problem in biotechnology and biomedicine: virus 2A and 2A-like sequences provide a solution

Luke, Garry A.; Ryan, Martin

doi:10.2217/fvl.13.82

Cited by 17 publications

(16 citation statements)

References 73 publications

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“…a) (Serra‐Soriano et al ., ). It is not known exactly how 2a peptides work, but they allow the splitting of the linked proteins during their translation (Luke and Ryan, ). Notably, because the MNSV(Al/GFP) RNA was larger than the native genome, it was not competent to be encapsidated.…”

Section: Resultsmentioning

confidence: 99%

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging, viral movement and interference with antiviral plant defence

Serra-Soriano

Navarro

Pallás

2016

Molecular Plant Pathology

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The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S-domain builds a robust protein shell around the viral genome, whereas the C-terminal protruding domain, or P-domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N-terminal domain, or R-domain, and the arm region, which connects the R-domain and S-domain, are involved in different key steps of the viral cycle, such as cell-to-cell movement and the suppression of RNA silencing and pathogenesis through their RNA-binding capabilities. Deletion mutants revealed that the CP RNA-binding ability was abolished only after complete, but not partial, deletion of the R-domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA-binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R-domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full-length CPs were required to encapsidate full-length genomes. We also found that the R-domain carboxyl portion and the arm region were essential for efficient cell-to-cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R-domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.

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Section: Resultsmentioning

confidence: 99%

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging, viral movement and interference with antiviral plant defence

Serra-Soriano

Navarro

Pallás

2016

Molecular Plant Pathology

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“…F2A is widely used for co‐expression, although other highly efficient “2A‐like” peptides have been identified and are also widely used in biotechnology and biomedicine: T2A from Thosea asigna virus, P2A from porcine teschovirus‐1 and E2A from equine rhinitis A virus (Figure ) . Many researchers employ a “spacer” sequence between the upstream protein and 2A: the V5 epitope tag: ‐GKPUPNPLLGLDST‐, a 3xFLAG epitope tag: ‐DYKDHDG‐DYKDHDI‐DYKDDDDK‐, or a glycine‐serine linker: ‐GSG‐ or ‐SGSG‐ …”

Section: Introductionmentioning

confidence: 99%

“…22 F2A is widely used for co-expression, although other highly efficient "2A-like" peptides have been identified and are also widely used in biotechnology and biomedicine: T2A from Thosea asigna virus, P2A from porcine teschovirus-1 and E2A from equine rhinitis A virus ( Figure 1). 29,30 Many researchers employ a "spacer" sequence between the upstream protein and 2A: the V5 epitope tag: 32 or a glycine-serine linker: -GSGor -SGSG-. 13,33 This technology has been critical for expression of heteromultimeric complexes and biochemical pathways in diverse areas such as human cancer gene therapies, production of induced pluripotent stem cells for regenerative medicine, creation of transgenic animals and plants, and the production of high-value proteins for the pharmaceutical industry.…”

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confidence: 99%

“Therapeutic applications of the ‘NPGP’ family of viral 2As”

Luke

Ryan

2018

Reviews in Medical Virology

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Oligopeptide "2A" and "2A-like" sequences ("2As"; 18-25aa) are found in a range of RNA virus genomes controlling protein biogenesis through "recoding" of the host-cell translational apparatus. Insertion of multiple 2As within a single open reading frame (ORF) produces multiple proteins; hence, 2As have been used in a very wide range of biotechnological and biomedical applications. During translation, these 2A peptide sequences mediate a eukaryote-specific, self-"cleaving" event, termed "ribosome skipping" with very high efficiency. A particular advantage of using 2As is the ability to simultaneously translate a number of proteins at an equal level in all eukaryotic systems although, naturally, final steady-state levels depend upon other factors-notably protein stability. By contrast, the use of internal ribosome entry site elements for co-expression results in an unbalanced expression due to the relative inefficiency of internal initiation. For example, a 1:1 ratio is of particular importance for the biosynthesis of the heavy-chain and light-chain components of antibodies: highly valuable as therapeutic proteins. Furthermore, each component of these "artificial polyprotein" systems can be independently targeted to different sub-cellular sites. The potential of this system was vividly demonstrated by concatenating multiple gene sequences, linked via 2A sequences, into a single, long, ORF-a polycistronic construct. Here, ORFs comprising the biosynthetic pathways for violacein (five gene sequences) and β-carotene (four gene sequences) were concatenated into a single cistron such that all components were co-expressed in the yeast Pichia pastoris. In this review, we provide useful information on 2As to serve as a guide for future utilities of this co-expression technology in basic research, biotechnology, and clinical applications.

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“…Under the monikers of "Skipping", "Stop-Carry On" and "StopGo" translation, it allows the stoichiometric production of multiple, discrete, protein products from a single transgene [23,24]. Several recent review articles have amply covered the role of 2A biotechnology in animal systems [20,25]. This summary-review will provide an up-to-date overview of 2A and cover the wider application of 2A-polyproteins to the expression of multiple proteins in plants.…”

Section: Introductionmentioning

confidence: 99%