|Title||Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolysis.|
|Publication Type||Journal Article|
|Year of Publication||1999|
|Authors||Pham, MQ, Naggie, S, Wier, M, Cha, HJ, Bentley, WE|
|Date Published||1999 Jan 20|
|Keywords||Animals, Drug Stability, Endopeptidases, Gene Expression, Humans, Interleukin-2, Larva, Moths, Nucleopolyhedrovirus, Protein Engineering, Protein Processing, Post-Translational, Recombinant Proteins, Temperature|
Many eukaryotic proteins have been successfully expressed in insect cells infected with a baculovirus in which the foreign gene has been placed under the control of a viral promoter. This system can be costly at large scale due to the quality of virus stock, problems of oxygen transfer, and severity of large-scale contamination. To circumvent this problem, we have investigated the expression of a foreign protein, human interleukin-2 (IL-2), in insect larvae, Trichoplusia ni, infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The IL-2 gene was placed under control of the p10 promoter so that the polyhedra remained intact for efficient primary infection. From our results, it was clear that early infection limited larval growth and late infection delayed product production until near pupation, hence infection timing was important. Also, the harvest time was crucial for obtaining high yield, because IL-2 production had a sharp optimal peak with a time of occurrence dependent on both temperature and the initial amount of infection virus. Specifically, we found that, by raising the infection temperature to 30 degrees C, we more than doubled the protein productivity. Furthermore, a significant concern of the larvae/baculovirus expression system has been the large amount of protease produced by the larvae, which adversely affects the protein yield. Therefore, we screened several protease inhibitors and characterized the larval protease specificity and timing to attenuate their impact. This report elucidates and delineates the factors that most directly impact protein yield in the larvae expression system, using IL-2 as a model.
|Alternate Journal||Biotechnol. Bioeng.|