Viral transduction of eukaryotic cell Recombinants is one possibility to generate recombinant proteins efficiently, and among all virus-based expression systems, the baculovirus/insect cell expression system (BEVS) is undoubtedly the best known and applied. This chapter describes the individual steps of the system process: maintenance of insect cell cultures, transfection of recombinant DNA into different insect cell lines, amplification of virus stocks, determination of virus titer, and complementary experiments necessary to generate conditions of optimal production.
Cell lines for the expression of recombinant proteins
Recombinant Protein Production In Mammalian And Insect Cell Lines
Due to their protein synthesis capabilities, eukaryotic cell lines have become essential for the process of recombinant protein production. Its ability to aid in complex post-translational modifications (glycosylation, phosphorylation), protein folding, and molecular assembly outperforms other systems. Recombinant protein production begins with expression vector engineering and transfection into the host system, followed by cell selection, cloning, screening and evaluation. To meet quality and scalability requirements, researchers producing recombinant proteins need efficient and cost-effective expression hosts.
Mammalian cells must often be used to produce recombinant proteins with the correct post-translational modifications. Recombinant proteins from mammalian cells have found use in therapeutics for diseases from diabetes to cancer, changing the landscape of medical care. This great potential has led to the exploration of various mammalian systems for hosting and secreting recombinant proteins. Currently, Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293) cells are preferentially used due to:
- High cell density in suspension culture and production of high protein yield
- Most successful transfection
- Survival in serum-free media
- Different strains available for targeted integration of transgenes
- Reduced risk of viral infection
Optimal mammalian cell lines are selected considering the productivity, bioactivity, purpose, and physicochemical characteristics of the protein of interest. Our mammalian cell lines support the detection of small-scale constructs as well as the rapid production of milligram-scale proteins.
Widely used for the production of recombinant proteins for structural studies, insect expression platforms are inexpensive, scalable, and less demanding than mammalian cells. These cells can be grown in spinner flasks or shakers and do not require a CO2-enriched atmosphere. Established insect cell lines can produce recombinant proteins in high yields and require smaller culture volumes.
Key Features of Insect Expression Platforms
- Signal peptides cleaved as in mammalian systems
- Disulfide bonds formed in the endoplasmic reticulum
- Proprotein converting enzymes available for proteolytic processing
- Membrane composition similar to mammalian systems
Media free of animal components
Traditional cell culture techniques require animal serum in concentrations up to 20% of the total culture volume. Based on concerns about extraneous agents sometimes found in animal-derived serum, serum-free media have become preferred in the production of recombinant protein for therapeutic use. Our offering of mammalian cell lines includes those that can be grown in media free of animal components.