High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

Falkner, Falko G.; Turecek, P. L.; MacGillivray, Ross T. A.; Bodemer, Walter; Scheiflinger, Friedrich; Kandels, S; Mitterer, Artur; Kistner, Otfried; Barrett, Noel; Eibl, Johann; Dorner, Friedrich

doi:10.1055/s-0038-1656335

Cited by 15 publications

(2 citation statements)

References 33 publications

(30 reference statements)

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“…A similar observation was reported by Arp et al, (5) who found that an increase in MOI from 2 to 10 did not significantly increase the yield of HTLV-1 gp46. Falkner et al (16) expressed prothrombin and Factor II in Vero, BHK, and HeLa cells by infection with a single recombinant vaccinia virus, and obtained yields for Factor II and prothrombin of 3-4 µg/ 10 6 cells and 1-4 µg/10 6 cells, respectively. They also determined the MOI effect in plates and found similar yields when MOI 0.1-10 were used.…”

Section: Resultsmentioning

confidence: 99%

Production of HIV-1 gp120 in Packed-Bed Bioreactor Using the Vaccinia Virus/T7 Expression System

Hu¹,

Kaufman²,

Cho

et al. 2000

Biotechnol. Prog.

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The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications. This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed-bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers.

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Section: Resultsmentioning

confidence: 99%

Production of HIV-1 gp120 in Packed-Bed Bioreactor Using the Vaccinia Virus/T7 Expression System

Hu¹,

Kaufman²,

Cho

et al. 2000

Biotechnol. Prog.

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“…For the expression of r-proteins in mammalian cells, baculovirus vectors have numerous advantages including: (1) the absence of viral replication, (2) the lack of cytotoxicity, (3) the technical simplicity of vector production, and (4) the large DNA insert capacity (up to 38 kb) allowing the delivery of multiple genes from a single vector (Kost et al 2000). Finally, vaccinia virus vectors have been used to produce rproteins such as nerve growth factor (Edwards et al 1988), Factor VIII (Pavirani et al 1987, and human pro-thrombin (Falkner et al 1992). The infectious nature of the virus is a major concern, and it requires containment in a Biosafety Level 2 laboratory.…”

Section: Viral Expression Vectorsmentioning

confidence: 99%

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

et al. 2007

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The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.

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Transient Expression Systems

Bernard¹,

Blasey²

1999

Encyclopedia of Bioprocess Technology

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High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

Cited by 15 publications

References 33 publications

Production of HIV-1 gp120 in Packed-Bed Bioreactor Using the Vaccinia Virus/T7 Expression System

Production of HIV-1 gp120 in Packed-Bed Bioreactor Using the Vaccinia Virus/T7 Expression System

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

Transient Expression Systems

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