Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide.

Marcos, José F.; Beachy, Roger N.

doi:10.1099/0022-1317-78-7-1771

Cited by 32 publications

(21 citation statements)

References 35 publications

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“…Dasgupta et al (1998) used the TVMV NIa proteinase to express two reporter genes (CAT and acetate kinase [AK]) in transgenic tobacco plants. As expected from the reports by von Bodman et al (1995) and Marcos and Beachy (1997), processed and enzymatically active CAT and AK could be observed in the resulting plant lines. Partial processing of a polyprotein bearing cytoplasmicallỳ targeted' CAT and AK was minimal in this report, suggestive of possible differences in processing efficiencies of different NIacontaining polyproteins.…”

Section: Strategies For Polycistronic or Polygenic Transgene Expressisupporting

confidence: 69%

“…It is possible, owing to differences in rates of cis-and trans-processing by embedded proteinases, that picornaviral-like proteinases may act inefficiently on polyproteins that possess large numbers of foreign (e.g., nonviral) polypeptides. It is also possible, as suggested by the contrasting processing efficiencies noted by Marcos and Beachy (1997) and Dasgupta et al (1998), that different proteins may fold or otherwise interact with the processing site or proteinase in a way that leads to less than quantitative processing of the expected polyprotein.…”

Section: Caveats and Prospects For The Use Of Multigene Expression Stmentioning

confidence: 94%

“…While an in planta demonstration of the accumulation of the processed forms of the polyprotein was not reported, in vitro processing was described, and the transgenic plant tissue did possess the enzyme activities expected from the successful expression of the polyproteinencoding gene. Marcos and Beachy (1997) utilized a similar strategy to express the tobacco mosaic virus (TMV) and soybean mosaic virus (SMV) coat proteins as a single polyprotein. In this case, the two coat protein (CP) genes were used in concert with the TEV NIa proteinase.…”

Section: Strategies For Polycistronic or Polygenic Transgene Expressimentioning

confidence: 99%

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Strategies for expressing multiple foreign genes in plants as polycistronic constructs

Hunt

Maiti

2001

In Vitro Cell.Dev.Biol.-Plant

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With an ever-increasing frequency, it has become desirable to express many foreign genes in the same transgenic plant. A range of approaches to this problem have been explored in the period since gene transfer was developed as a useful tool in plant science. These have ranged from assembly of multiple transcription units on a single DNA vector, to the execution of several independent transformations involving single genes followed by successive rounds of hybridization to yield plant lines with the desired combination of genes, to cotransformation with multiple plasmids followed by molecular screening to identify desired transgenic plants. Recent developments have provided new alternatives to the problem of the expression of multiple foreign genes in plants, approaches that revolve around the expression of polycistronic mRNAs. This review summarizes the current status of the area of polycistronic gene expression in transgenic plants, within the context of multigene expression.

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Section: Strategies For Polycistronic or Polygenic Transgene Expressisupporting

confidence: 69%

Section: Caveats and Prospects For The Use Of Multigene Expression Stmentioning

confidence: 94%

Section: Strategies For Polycistronic or Polygenic Transgene Expressimentioning

confidence: 99%

See 1 more Smart Citation

Strategies for expressing multiple foreign genes in plants as polycistronic constructs

Hunt

Maiti

2001

In Vitro Cell.Dev.Biol.-Plant

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“…It has been shown that the protein cloned at the C-terminus was produced in a lower amount than the N-terminal protein. Marcos and Beachy (1997), mentioned possible premature termination of translation as a mechanism for imbalance. The reason may be the nuclear localization signals (NLSs) present in the NIa, targeting the polyprotein to the nucleus.…”

Section: Proteolytic Processingmentioning

confidence: 97%

Polycistronic Viral Vectors

Felipe

2002

CGT

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Traditionally, vectors for gene transfer/therapy experiments were mono- or bicistronic. In the latter case, vectors express the gene of interest coupled with a marker gene. An increasing demand for more complex polycistronic vectors has arisen in recent years to obtain complex gene transfer/therapy effects. In particular, this demand is stimulated by the hope of a more powerful effect from combined gene therapy than from single gene therapy in a process whose parallels lie in the multi-drug combined therapies for cancer or AIDS. In the 1980's we had only splicing signals and internal promoters to construct such vectors: now a new set of biotechnological tools enables us to design new and more reliable bicistronic and polycistronic vectors. This article focuses on the description and comparison of the strategies for co-expression of two genes in bicistronic vectors, from the oldest to the more recently described: internal promoters, splicing, reinitiation, IRES, self-processing peptides (e.g. foot-and-mouth disease virus 2A), proteolytic cleavable sites (e.g. fusagen) and fusion of genes. I propose a classification of these strategies based upon either the use of multiple transcripts (with transcriptional mechanisms), or single transcripts (using translational/post-translational mechanisms). I also examine the different attempts to utilize these strategies in the construction of polycistronic vectors and the main problems encountered. Several potential uses of these polycistronic vectors, both in basic research and in therapy-focused applications, are discussed. The importance of the study of viral gene expression strategies and the need to transfer this knowledge to vector design is highlighted.

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“…Alternatively, incorporating an exogenous protease within the polyprotein system is used to overcome this problem. Some notable examples include polyprotein vectors that employ NIa protease recognition sequence together with the Nla proteinase from the tobacco etch virus (Marcos and Beachy, 1994, 1997) and a similar vector that employs a NIa proteinase from the tobacco vein mottling virus (Dasgupta et al ., 1998). While proper polyprotein processing was achieved using these vectors, the protein expression levels were generally low.…”

Section: Introductionmentioning

confidence: 99%

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

Zhang

Rapolu

Kumar³

et al. 2017

Plant Biotechnology Journal

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SummaryA novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage is covalently linked to the foot‐and‐mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an ‘IntF2A’ self‐excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a ‘polyprotein’ precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C‐terminal F2A extension remains on the released POIs. We demonstrated co‐expression of as many as three proteins in plants without compromising expression levels when compared with those using single‐protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti‐His Tag antibody in N. benthamiana leaves. The IntF2A‐based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co‐expression in plants, which is particularly important for gene/trait stacking.

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Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide.

Cited by 32 publications

References 35 publications

Strategies for expressing multiple foreign genes in plants as polycistronic constructs

Strategies for expressing multiple foreign genes in plants as polycistronic constructs

Polycistronic Viral Vectors

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

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