Abstract
Recombinant human granulocyte colony-stimulating factor (G-CSF) produced in cultured mammalian cells undergoes proper posttranslational modifications and, thereby, possesses better pharmacological properties in comparison with the homologous protein expressed in bacteria. Biopharmaceuticals derived from cell culture tend to be expensive because of lower yields compared to bacterially expressed competitors and numerous issues with the scalability of production. Particular limitation of scalability pertains to delivery of expression vectors to the cell culture. Natural and efficient way to deliver foreign DNA or RNA to cell is a viral infection. We intended to develop the viral genome capable of G-CSF expression.Objectives: To develop autonomously replicating viral RNA capable of heterologous expression of the G-CSF in cultured mammalian cells.Methods: Genetic engineering, cell culture, and virology.Results: Viable genome of the Venezuelan equine encephalomyelitis virus (VEE) was constructed. The G-CSF gene was synthesized de novo. Gene cassette GFP-2A-G-CSF was constructed for simultaneous expression of the fluorescent marker of viral replication (GFP) and the product of interest. Recombinant viral genome VEE/GFP-2A-G-CSF was assembled; its viability was confirmed upon transfection to the BHK-21 cells.Conclusion: Genetic engineering of the cDNA copies of the genomes of RNA viruses provides excessive opportunities to develop mammalian expression systems. We achieved production of recombinant proteins in cultured mammalian cells transfected with the VEE viral RNA carrying the inserted genes of marker protein and G-CSF.
Highlights
In the production of biopharmaceuticals, recombinant pharmacologically active proteins became an important segment of high-tech economy with great potential for further expansion
We achieved production of recombinant proteins in cultured mammalian cells transfected with the VEE viral RNA carrying the inserted genes of marker protein and granulocyte colony-stimulating factor (G-CSF)
We describe the VEE genome designed for transient simultaneous expression of two proteins, G-CSF and a fluorescent marker (GFP) in infected or transfected cell lines
Summary
In the production of biopharmaceuticals, recombinant pharmacologically active proteins became an important segment of high-tech economy with great potential for further expansion. Some valuable proteins cannot be produced in a biologically active form in bacteria. These include, among others, the erythropoietin and the anti-hemophilic factor VIII, which are produced for medical use in the mammalian cell cultures. One form of the G-CSF produced in industrial scales in the E.coli is present in the market with an international non-proprietary name (INN) “Filgrastim” and a large number of patented brand marks. Different form of G-CSF is produced in the in vitro cultured CHO cells, which are transformed with the eukaryotic expression plasmid. This kind of G-CSF has INN “Lenograstim,” whereas Filgrastim lacks glycosylation and bears non-natural formylmethionine residue at the N-terminus. Lenograstim has greater potency compared to Filgrastim, probably because it is more stable to proteolysis (Ataergin et al, 2008)
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