| The baculovirus expression vector (BEV) system was employed for expression of recombinant proteins and as a tool for monitoring the infection in Spodoptera frugiperda-9 (Sf-9) insect cells and Trichoplusia ni (T. ni) insect larvae. First, the BEV system was used to express a fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) in suspended Sf-9 insect cells, which was then successfully purified. The fusion was comprised of a histidine affinity ligand to simplify purification by using an immobilized metal affinity chromatography (IMAC) column, GFP as a marker, and an enterokinase cleavage site for recovery of hIL-2. Results here demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in suspended insect cells.; For monitoring the infection progress in insect cells and insect larvae, a BEV construct was made which expressed GFPuv under the control of the Early-to-Late (ETL) baculovirus promoter. This construct resulted in expression of GFP approximately 18 hours earlier in suspended insect cell cultures and approximately 10 hours earlier in insect larvae. Importantly, in suspended insect cell cultures, GFPuv was expressed earlier under the ETL promoter, and the level of fluorescence also indicated the relative level of expression of the model product protein. Results here demonstrated that GFP was a rapid, non-invasive marker of gene expression and protein production in both host systems.; Lastly, two strong, very late baculovirus promoters were used to express hIL-2 either as a fusion with GFPuv or alone as a single protein. Here, it was shown that GFP enabled clear indication of the infection process and more importantly, the quantity of model product under both promoters. Although the profiles of the two promoter systems were remarkably similar, the baculovirus with the genes present under p10 control produced relatively more recombinant protein in larvae. |
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