Identification of biotinylated molecules using a baculovirus-expressed luciferase-streptavidin fusion protein.
A genetic fusion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus plagiophthalamus was constructed. The fusion protein was produced in the Sf9 insect cell line using the baculovirus expression vector system (BEVS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins from cells infected with the recombinant virus, VL1393-LucGR-StreptAv, revealed that the fusion protein migrated with an apparent molecular weight of 75 kDa. Light emission measurements showed that the infected cells produced about 255 mg of the chimeric protein per liter of cell culture (127.5 micrograms/1 x 10(6) cells). Precipitation of the LucGR-StreptAv fusion protein with biotinylated acrylic beads as well as immunoblot analyses using biotinylated immunoglobulins indicated that both fusion moieties of the chimeric protein product were functional with respect to their physical and enzymatic activities.
Karp
M
M
University of Turku, Finland.
Lindqvist
C
C
Nissinen
R
R
Wahlbeck
S
S
Akerman
K
K
Oker-Blom
C
C
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting / Q000379:methods*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008163:Luminescent Measurements; D008863:Microspheres; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D019809:Streptavidin
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D008156:Luciferases