Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus
- 94 Downloads
Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×107 cells.ml−1, and a β-gal concentration of up to 104,000 unit.ml−1 were achieved.
Key wordsinsect cells media comparison MOI protein expression scale-up temperature-shift
Unable to display preview. Download preview PDF.
- Brown M and Faulkner P (1978) Plaque assay of nuclear polyhedrosis viruses in cell culture. Appal. Environm. Microbiol. 36: 31–35.Google Scholar
- Hink WF and Strauss E (1976) Growth of theTrichoplusia ni (TN-368) cell line in suspension culture. In: Kurstak E and Maramorosch K (eds.) Invertebrate Tissue Culture: Applications in Medicine, Biology, and Agriculture. Academic Press, New York, pp. 297–300.Google Scholar
- King G, Kuzio J, Daugulis A, Faulkner P, Alien B, Wu J and Goosen M (1991) Assessment of virus production and chloramphenicol acetyl transferase expressed by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus. Biotechnol. Bioeng. 38: 1091–1099.CrossRefGoogle Scholar
- Luckow VA (1991) Cloning and expression of heterologous genes in insect cells with baculovirus vectors. In: Prokop A, Bajpai RK and Ho CS (eds.) Recombinant DNA Technology and Applications. McGraw-Hill, Inc., New York, pp. 97–152.Google Scholar
- Maniatis T, Fritsch EF and Sambrook J (1982) Molecular Cloning, 1st edn. Cold Spring Harbor, NY.Google Scholar
- Miller JH (1972) Experiments in molecular genetics Cold Spring Harbor Laboratory, New York.Google Scholar
- Neumann JR, Morency CA and Russian KS (1987) A novel rapid assay for chloramphenicol acetyl transferase gene expression. Biotechniques 5 (5): 4440–450.Google Scholar
- Weiss SA, Belisle BW, DeGovanini A, Godwin G, Kohler J and Summers MD (1989) Insect cells as substrates for biologicals. Develop. Biol. Standard. 70: 271–279.Google Scholar
- Weiss SA, Gorfien S, Fike R, Disorbo D and Dayme D (1990) Large-scale production of proteins using serum-free insect cell culture. Presented at the Ninth Australian Biotechnology Conference, Cold Coast, Queensland, Australia.Google Scholar