An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

doi:10.1007/s13238-012-2101-y

Cited by 6 publications

(4 citation statements)

References 27 publications

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“…The protease domain of the NIa protein of PPV has demonstrated a notable biotechnological interest as its high efficiency and specificity make it very attractive for the processing of fusion proteins both in vitro (Pérez‐Martín et al ., ; Zheng et al ., ) and in vivo (Zheng et al ., ).…”

Section: Ppv As a Tool In Biotechnologymentioning

confidence: 97%

Plum pox virus and sharka: a model potyvirus and a major disease

Garcı́a

Glasa

Cambra

et al. 2014

Molecular Plant Pathology

195

151

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A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic plum, 'HoneySweet', transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.

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Section: Ppv As a Tool In Biotechnologymentioning

confidence: 97%

Plum pox virus and sharka: a model potyvirus and a major disease

Garcı́a

Glasa

Cambra

et al. 2014

Molecular Plant Pathology

195

151

View full text Add to dashboard Cite

show abstract

“…Although both proteins were produced, only poor expression was obtained (not shown). We therefore chose an alternative strategy based on expression of cleavable polyproteins that have been successfully produced after expression in E. coli [ 17 , 18 ], in insect cells infected with baculovirus vectors [ 19 , 20 ] and in mammalian cells [ 21 , 22 ]. The genes encoding two or more proteins known to assemble into a complex were fused together into a single open reading frame downstream of an IPTG inducible T7 promoter.…”

Section: Resultsmentioning

confidence: 99%

Efficient production of protein complexes in mammalian cells using a poxvirus vector

et al. 2022

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The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10–50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

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“…Although both proteins were produced, only poor expression was obtained (not shown). We therefore chose an alternative strategy based on expression of cleavable polyproteins that have been successfully produced after expression in E. coli [14,15], in insect cells infected with baculovirus vectors [16,17] and in mammalian cells [18,19]. The genes encoding several proteins known to assemble into a complex were fused together into a single open reading frame downstream of an IPTG inducible T7 promoter.…”

Section: The Polyprotein Precursor Strategy As Applied To Two Hiv Pro...mentioning

confidence: 99%

Efficient production of protein complexes in mammalian cells using a poxvirus vector

Drillien¹,

Pradeau‐Aubreton²,

Batisse³

et al. 2022

Preprint

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Background: The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Results: Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. Conclusions: The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

show abstract

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

Cited by 6 publications

References 27 publications

Plum pox virus and sharka: a model potyvirus and a major disease

Plum pox virus and sharka: a model potyvirus and a major disease

Efficient production of protein complexes in mammalian cells using a poxvirus vector

Efficient production of protein complexes in mammalian cells using a poxvirus vector

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