Highly efficient production of GFP and its derivatives in insect cells for visual in vitro applications.
We have generated recombinant baculoviruses for expression of the green fluorescent protein (GFP), a bright GFP mutant (S65T), and a GFP-streptavidin fusion protein in Sf9 and High Five insect cell lines. At 3-4 days post infection, about 30% of the total protein contents was represented by the recombinant protein products, giving the infected insect cells a bright green color which was clearly visible by eye in daylight. The isolated GFP-streptavidin fusion protein, which possessed fluorescence properties identical to those of the native GFP, was capable of binding biotin as shown by using biotinylated beads as well as biotinylated antibody complexes decorating surface expressed GluR-6 glutamate receptor in live and fixed insect cells. The exceptionally high expression levels of GFP and GFP (S65T) and the GFP-streptavidin fusion protein in recombinant baculovirus infected insects should facilitate production of GFP derivatives for in vitro applications.
Oker-Blom
C
C
VTT Biotechnology and Food Research, Espoo, Finland.
Orellana
A
A
Keinanen
K
K
D000818:Animals; D001426:Bacterial Proteins; D016367:Baculoviridae / Q000235:genetics*; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D007313:Insecta; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009036:Moths; D011485:Protein Binding; D017470:Receptors, Glutamate / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin
D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D017470:Receptors, Glutamate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001710:Biotin; D019809:Streptavidin