Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone.

Abstract

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng. 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids, glucose and yeastolate ultrafiltrate) into Sf 9 cell cultures grown in SF 900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 microg/ml of NS1 at a peak cell density of 4.2 x 10 (6) cells/ml. In contrast, optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 microg/ml of NS1 produced at a peak cell density of 11.3 x 10 (6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10 (6) cells/ml, unless the cultures were stimulated by the addition of 4 microg/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 microg/ml, which was nearly 5-fold higher than optimised batch cultures.

• Authors: Leslie C L Chan, Paul R Young, Cheryl Bletchly, Steven Reid
• Journal: Journal of virological methods (J Virol Methods) Vol. 105 Issue 1 Pg. 87-98 (Aug 2002) ISSN: 0166-0934 [Print] Netherlands
• PMID: 12176145 (Publication Type: Evaluation Study, Journal Article)

Chemical References

• Culture Media
• NS1 protein, Dengue virus type 2
• Recombinant Proteins
• Viral Nonstructural Proteins
• Ecdysterone
• beta-Galactosidase

Topics

• Animals
• Cell Line
• Culture Media
• Dengue Virus (genetics)
• Ecdysterone (metabolism)
• Genetic Vectors
• Nucleopolyhedroviruses (genetics, metabolism)
• Recombinant Proteins (genetics, metabolism)
• Spodoptera
• Viral Nonstructural Proteins (genetics, metabolism)
• beta-Galactosidase (metabolism)