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Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript.
Gorben P Pijlman1, Els C Roode, Xiaoxiang Fan
1Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
Journal of Biotechnology
|December 21, 2005
Summary
A new baculovirus vector stabilizes recombinant protein production in insect cells by preventing yield loss during scale-up. This novel system enhances protein expression through continued virus passages, crucial for bioreactor applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Virology
Background:
- Recombinant protein production in insect cells using baculovirus vectors faces challenges with decreasing yields over time, known as the passage effect.
- This passage effect is caused by defective interfering baculoviruses (DIs) that hinder the replication of functional viruses.
Purpose of the Study:
- To develop a novel baculovirus expression vector that overcomes the passage effect and improves recombinant protein yield stability.
- To enhance the efficiency of large-scale recombinant protein production in insect-cell bioreactors.
Main Methods:
- A bicistronic expression cassette was engineered into a baculovirus vector for simultaneous expression of a recombinant gene (GFP) and an essential viral gene (GP64).
- An internal ribosome entry site (IRES) from Rhopalosiphum padi virus (RhPV) was used to drive GP64 translation, with the native GP64 gene deleted.
- This design creates a selection pressure for maintaining the bicistronic mRNA, thereby stabilizing foreign gene expression.
Main Results:
- The novel bicistronic vector demonstrated significantly higher and more stable GFP expression compared to a control vector across multiple virus passages.
- The stabilized vector showed versatility by successfully propagating in various insect cell lines, including Sf21, Sf9, and High Five cells.
Conclusions:
- The developed bicistronic baculovirus vector effectively mitigates the passage effect, ensuring consistent recombinant protein production.
- This stabilized vector is highly valuable for large-scale recombinant protein manufacturing in insect-cell bioreactors, particularly in processes requiring numerous viral passages.