Microbial Cell Factories

, 5:P12 | Cite as

RNA Interference mediated knockdown of genes in order to increase protein production using the baculovirus expression system

  • Colin Hebert
  • Eun Jeong Kim
  • Shannon F Kramer
  • James J Valdes
  • William E Bentley
Open Access
Poster Presentation
  • 3.9k Downloads

Keywords

Green Fluorescent Protein Recombinant Baculovirus Baculovirus Expression System Reduce Cell Death Infected Cell Culture 

Background

The baculovirus expression system has proven to be a robust and versatile system for recombinant protein production in insect cells. A wide range of promoters is available for the facile expression of transgenes, and yields of up to 50% of total protein have been reported [1]. However, in many cases yield is decreased as a result of proteases and host cell apoptosis [2]. Past efforts to overcome this problem include co-expressing chaperone proteins to assist with folding [3], anti-apoptotic proteins to reduce cell death, or adding chemical protease inhibitors to the culture media [4]. However, these methods may have non-specific effects, prove too costly to be practical, or impose an undue metabolic burden on an already stressed cell. An alternative approach to increasing protein production is through the application of RNA interference (RNAi) to knockdown viral and host genes responsible for decreasing the yield of recombinant protein. Potential targets include proteases, cell-death proteins, and cell cycle regulators. By altering the metabolic landscape of cells prior to the introduction of the baculovirus, protein production can be improved.

Results

Initially, double stranded RNA (dsRNA) was produced in vitro against 19 gene targets; including acidic juvenile hormone-sensitive protein (AJHSP1) and Elongation factor 2 in Trichoplusia ni (T. ni) larvae, and a virus-encoded cathepsin-like protease, v-cath, in Spodoptera frugiperda (Sf-9) cell culture. The knockdown of these three genes increased the yield of Green Fluorescent Protein (GFP) produced via recombinant baculovirus. In the case of v-cath, the increase in infected cell culture was ~3-fold (Figure 1). Coincident with GFP yield, v-cath dsRNA also prolonged cell viability by over a day in infected Sf-9 cells (Figure 1). Ongoing studies are focused on identifying new host cell gene targets in both Sf-9 and T. ni cell culture, investigating combinations of knockdown targets, and integrating the most promising targets into expression vectors for the production of in vivo RNAi.
Figure 1

Effect of silencing of v-cath on the specific growth rate (A), the viability (B), and GFP production (C) in baculovirus infected Sf-9 cells. Cell counts were measured using a hemocytometer and viable cells were detected using trypan blue. Cell viability was defined by the ratio of the viable cell number to the total cell number. (◆) cells treated without dsRNA; (■) cells treated with dsRNA. GPF was measured quantitatively (C) using a fluorescence spectrometer. (black bar) cells without dsRNA; (white bar) cells treated with dsRNA.

Conclusion

Overall, our results support the application of RNAi as a metabolic engineering tool, specifically for enhancing protein productivity in the baculovirus expression system.

References

  1. 1.
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    Kost TA, Condreay JP, Jarvis DL: Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol. 2005, 23: 567-575. 10.1038/nbt1095.CrossRefGoogle Scholar
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    Ailor E, Betenbaugh MJ: Modifying secretion and post-translational processing in insect cells. Curr Opin Biotechnol . 1999, 10: 142-145. 10.1016/S0958-1669(99)80024-X.CrossRefGoogle Scholar
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    Kato T, Murata T, Usui T, Park EY: Improvement of GFPuv-beta 3GnT2 fusion protein production by suppressing protease in baculovirus expression system. Biosci Biotechnol Biochem. 2003, 67: 2388-2395. 10.1271/bbb.67.2388.CrossRefGoogle Scholar

Copyright information

© Hebert et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

Authors and Affiliations

  • Colin Hebert
    • 1
  • Eun Jeong Kim
    • 1
  • Shannon F Kramer
    • 2
  • James J Valdes
    • 3
  • William E Bentley
    • 1
    • 4
  1. 1.Center for Biosystems ResearchUniversity of Maryland Biotechnology InstituteCollege ParkUSA
  2. 2.Department of Biomedical Sciences, Baylor College of DentistryTexas A&M University System Health Science CenterDallasUSA
  3. 3.U. S. Army Edgewood Chemical Biological CenterAberdeen Proving GroundMarylandUSA
  4. 4.Department of Chemical and Biomolecular EngineeringUniversity of Maryland College ParkCollege ParkUSA

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