Expression of functional full-length hSRC-1 in eukaryotic cells using modified vaccinia virus Ankara and baculovirus

Ősz, Judit; Pradeau‐Aubreton, Karine; Drillien, Robert; Troffer-Charlier, N.; Kolb-Cheynel, Isabelle; Poterszman, Arnaud; Ruff, Marc; Moras, Dino; Rochel, Natacha

doi:10.1016/j.ab.2012.04.006

Cited by 2 publications

(4 citation statements)

References 11 publications

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“…In prior studies we have used adherent BHK 21 cells for protein production after infection with MVA recombinants [ 1 , 2 , 5 ]. However, adherent cells are not practical for handling large cell volumes particularly for expression and recovery of proteins that remain intracellular as is the case for the gene products we have expressed so far.…”

Section: Resultsmentioning

confidence: 99%

“…coli lac operator and lac repressor switch under the control of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) [ 3 , 4 ]. Using this strategy our laboratory has overexpressed and purified several full length cellular and viral proteins for functional and structural studies [ 5 – 8 ]. Despite its efficiency, the current MVA expression system still has a number of drawbacks such as the time required to isolate virus recombinants, the difficulty to scale up protein production and to co-express multiple proteins belonging to a protein complex upon infection with a single virus.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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show abstract

“…In prior studies we have used adherent BHK 21 cells for protein production after infection with MVA recombinants [1,2,5]. However, adherent cells are not practical for handling large cell volumes particularly for expression and recovery of proteins that remain intracellular as is the case for the gene products we have expressed so far.…”

Section: Protein Production In Suspension Cell Culturesmentioning

confidence: 99%

“…Gene expression from this viral vector is tightly regulated by an E. coli lac operator and lac repressor switch under the control of the inducer Isopropyl β-D-1thiogalactopyranoside (IPTG) [3,4]. Using this strategy our laboratory has overexpressed and puri ed several full length cellular and viral proteins for functional and structural studies [5][6][7][8]. Despite its e ciency, the current MVA expression system still has a number of drawbacks such as the time required to isolate virus recombinants, the di culty to scale up protein production and to co-express multiple proteins belonging to a protein complex upon infection with a single virus.…”

Section: Introductionmentioning

confidence: 99%

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

Drillien¹,

Pradeau‐Aubreton²,

Batisse³

et al. 2022

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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.

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Expression of functional full-length hSRC-1 in eukaryotic cells using modified vaccinia virus Ankara and baculovirus

Cited by 2 publications

References 11 publications

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

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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