Expression of green fluorescent protein in insect larvae and its application for heterologous protein production.
Many eukaryotic proteins have been successfully expressed in insect cells infected with a recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). There are, however, disadvantages with this cell-based system when carried out in suspension cultures at high bioreactor volume (e.g., limited oxygen transfer, susceptibility to contamination, high cost). These problems can be avoided by using whole larvae as the "reactors." There are, however, other problems encountered with larvae, one being their inaccessibility for product sampling. To combat this problem, we have investigated the expression of green fluorescent protein (GFP) as a reporter molecule in Trichoplusia ni insect larvae. A high production level of GFPuv (1.58 mg per larva, 26% of total protein) was obtained, enabling the rapid and non-invasive monitoring of GFP. Bright green light was emitted directly from the large opaque carcasses ( approximately 30mm) after illumination with UV light. Based on the green light intensity and a correlation between intensity and GFP mass, we determined the optimal harvest time (c.a. approximately 3 days post-infection). In parallel experiments, we expressed human interleukin-2 (IL-2) from another recombinant baculovirus with an almost identical expression profile. Since both GFP and IL-2 were rapidly degraded by protease activity during the fourth day post-infection (another disadvantage with larvae), we found an accurate determination of harvest time was critical. Correspondingly, our results demonstrated that GFP was an effective on-line marker for expression of heterologous protein in insect larvae.
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Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA.
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Rao
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Bentley
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