EXPRESSION OF GREEN FLUORESCENT PROTEIN IN INSECT LARVAE AND ITS APPLICATION FOR HETEROLOGOUS PROTEIN-PRODUCTION

Many eukaryotic proteins have been successfully expressed in insect ce lls infected with a recombinant baculovirus derived from the Autograph a californica nuclear polyhedrosis virus (AcNPV). There are, however, disadvantages with this cell-based system when carried out in suspensi on cultures at high bioreactor volume (e.g., limited oxygen transfer, susceptibility to contamination, high cost). These problems can be avo ided by using whole larvae as the ''reactors.'' There are, however, ot her 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 rapi d and non-invasive monitoring of GFP. Bright green light was emitted d irectly from the large opaque carcasses (similar to 30mm) after illumi nation with UV light. Based on the green light intensity and a correla tion between intensity and GFP mass, we determined the optimal harvest time (c.a. similar to 3 days post-infection). In parallel experiments , we expressed human interleukin-2 (IL-2) from another recombinant bac ulovirus with an almost identical expression profile. Since both GFP a nd IL-2 were rapidly degraded by protease activity during the fourth d ay post-infection (another disadvantage with larvae), we found an accu rate determination of harvest time was critical. Correspondingly, our results demonstrated that GFP was an effective on-line marker for expr ession of heterologous protein in insect larvae. (C) 1997 John Wiley & Sons, Inc.