E unum pluribus: multiple proteins from a self-processing polyprotein

Felipe, Pablo de; Luke, Garry A.; Hughes, Lorraine E.; Gani, David; Halpin, Claire; Ryan, Martin D.

doi:10.1016/j.tibtech.2005.12.006

Cited by 328 publications

(319 citation statements)

References 52 publications

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“…One of the most promising approaches has taken advantage of strategies that single-strand RNA viruses use to mediate multigene expression. Picornaviruses use 2A peptides, which function as cis-acting hydrolase elements (CHYSELs), to mediate 'cleavage' between two proteins [2][3][4] . These sequences were first discovered in the foot-and-mouth disease virus (FMDV) 5 , which encodes a single, long open reading frame in which two of the gene products are separated by the short 2A sequence.…”

Section: Multicistronic Vectors Using 2a Peptidesmentioning

confidence: 99%

Generation of T-cell receptor retrogenic mice

et al. 2006

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Section: Multicistronic Vectors Using 2a Peptidesmentioning

confidence: 99%

Generation of T-cell receptor retrogenic mice

et al. 2006

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“…The process was originally termed 'cleavage' by analogy with the proteasemediated cleavages occurring at other sites in the FMDV polyprotein but this is misleading because the 'cleavage' results from failure to form a peptide bond during translation. Unlike reinitiation of translation by IRESs, skipping induced by 2A sequences gives approximately equal expression of the proteins upstream and downstream of the 2A site (de Felipe et al, 2006). 2A and 2A-like sequences have previously been used in biotechnology applications, but not in adenoviruses (reviewed by de Felipe et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…Unlike reinitiation of translation by IRESs, skipping induced by 2A sequences gives approximately equal expression of the proteins upstream and downstream of the 2A site (de Felipe et al, 2006). 2A and 2A-like sequences have previously been used in biotechnology applications, but not in adenoviruses (reviewed by de Felipe et al, 2006). Protein IX is a small cement protein located between the hexons in the capsid of the adenovirus (Furcinitti et al, 1989).…”

mentioning

confidence: 99%

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Funston

Kallioinen

Felipe

et al. 2008

Journal of General Virology

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Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the junction of the 2A and downstream sequences, leading to the production of two proteins from a single open reading frame. Adenoviral protein IX is a minor capsid protein that has been used to display foreign peptides on the surface of the capsid. We have used 2A sequences from the foot-and-mouth disease virus (FMDV) and porcine teschovirus 1 (PTV-1) to express protein IX (pIX) and green fluorescent protein (GFP) from pIX-2A-GFP fusion genes in an oncolytic virus derived from human adenovirus 5. GFP was efficiently expressed by constructs containing either 2A sequence. Peptide bond skipping was more efficient with the 58 aa FMDV sequence than with the 22 aa PTV-1 2A sequence, but the virus with the FMDV 2A sequence showed a reduction in plaque size, cytopathic effect, viral burst size and capsid stability. We conclude that ribosome skipping induced by 2A sequences is an effective strategy to express heterologous genes in adenoviruses; however, careful selection or optimization of the 2A sequence may be required if protein IX is used as the fusion partner. INTRODUCTIONReplication-competent oncolytic adenoviruses have been extensively tested in animals, and a virus lacking the E1B 55K gene was recently approved for cancer therapy in China (reviewed by Alemany, 2007). The main factor limiting widespread application of oncolytic adenoviruses is their low efficacy. Many groups have attempted to develop more active viruses, for example by inserting genes for toxic proteins or prodrug-activating enzymes (reviewed by Alemany, 2007). We and others have previously used internal ribosome entry sites (IRESs) to initiate the translation of prodrug-activating enzymes in adenoviruses but the results were disappointing (reviewed by de Felipe, 2002;Fuerer & Iggo, 2004;Lukashev et al., 2005). Ribosome skipping is a recently described mechanism that allows translation of multiple proteins from a single mRNA. It is based on the use of a picornaviral 2A sequence that causes the ribosome to continue translation after skipping the formation of one peptide bond. This process was first characterized in foot-and-mouth disease virus (FMDV) (Ryan & Drew, 1994). The process was originally termed 'cleavage' by analogy with the proteasemediated cleavages occurring at other sites in the FMDV polyprotein but this is misleading because the 'cleavage' results from failure to form a peptide bond during translation. Unlike reinitiation of translation by IRESs, skipping induced by 2A sequences gives approximately equal expression of the proteins upstream and downstream of the 2A site (de Felipe et al., 2006). 2A and 2A-like sequences have previously been used in biotechnology applications, but not in adenoviruses (reviewed by de Felipe et al., 2006). Protein IX is a small cement protein located between the hexons in the capsid of the adenovirus (Furcinitti et al., 1989). It is essential for the packaging of full-length viral genomes (Ghosh-Cho...

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“…This result suggests that a target gene should be fused to the gfp gene in order to be positively correlated with the expression of both genes. To achieve separate expression of the target protein, the inclusion of an internal ribosome entry sites (IRES) that would allow cap-independent translation initiation, or 2A oligopeptides that mediate self-cleavage at the C-terminus that could allow efficient separation of GFP and the recombinant protein, are strategies that could be considered (de Felipe et al 2006;Matsuo et al 2004;Stewart 2005;Urwin et al 2000). There is a positive association between estimated GFP protein concentration and GFP fluorescence (R 2 = 0.82, Fig.…”

Section: Discussionmentioning

confidence: 99%

Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products

Saika

Toki

2009

Plant Cell Rep

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In genetic transformation systems, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. However, simultaneous use of the gene encoding green fluorescent protein (GFP) and an antibiotic resistance gene facilitates the selection process, since it allows visible selection of transformed cells. Here, we report the development of a visual selection system for transformed cells using a GFP marker without selection against antibiotics after Agrobacterium-mediated transformation in rice. Both GFP protein levels and GFP fluorescence in calli isolated by visual selection were higher than in calli selected on hygromycin (Hyg), suggesting that transgenic calli hyper-accumulating GFP were efficiently obtained by selection using GFP fluorescence itself rather than Hyg resistance. Furthermore, gfp transcripts in calli isolated by visual selection were more abundant than under Hyg selection; in contrast, transcript levels of hpt in calli selected visually were comparable to those obtained under Hyg selection. These results suggest that there was no correlation between hpt and gfp expression levels, despite the fact that they are aligned in tandem on an integrated locus after selection by either GFP fluorescence or Hyg resistance. This fact indicates that positional effects can influence the expression of each transgene differently, even when they are located in tandem at the same locus. In summary, based on our results, we discuss a model system for rice cell culture transformation for the production of recombinant proteins using visual selection.

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E unum pluribus: multiple proteins from a self-processing polyprotein

Cited by 328 publications

References 52 publications

Generation of T-cell receptor retrogenic mice

Generation of T-cell receptor retrogenic mice

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products

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