Maximizing production of estrogen receptor beta with the baculovirus expression system.
Steroid hormone/nuclear receptor expression in cultured insect cell lines is routinely driven by a baculovirus vector. An advantage of the baculovirus production of these receptors is that large amounts of functional receptors are obtained for subsequent in vitro studies. Most laboratories produce nuclear receptors in Spodoptera frugiperda (Sf)9 cells. However, no one has determined whether this cell line is optimal for the production of any nuclear receptor. We compared the time course and level of estrogen receptor beta (ER beta) production from a baculovirus in two S. frugiperda cell lines, IPLB-SF21AE (Sf21) and Sf9, and two Trichloplusia ni cell lines, Tn368 and BTI-TN5b1-4 (High Five). Cells were harvested at various times (0.5-5 days) after infection. ER beta expression and activity was determined by specific [3H]estradiol (E2) binding, Western blot analysis, and estrogen response element (ERE) binding in vitro. The highest functional, bioactive ER beta expression both at the earliest time after infection and in the amount of ER beta produced/cell was with the Sf21 cell line. Baculovirus expressed ER beta-bound EREs with high affinity in a DNA sequence-dependent manner. We conclude that Sf21 cells are the best-suited cells for ER beta production.
Kulakosky
Peter C
PC
University of Louisville School of Medicine, Louisville, KY, USA.
Klinge
Carolyn M
CM
D000818:Animals; D016367:Baculoviridae / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D047629:Estrogen Receptor beta; D007915:Lepidoptera / Q000145:classification / Q000235:genetics / Q000821:virology; D011786:Quality Control; D051381:Rats / Q000235:genetics; D011960:Receptors, Estrogen / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013045:Species Specificity; D018411:Spodoptera / Q000145:classification / Q000235:genetics / Q000821:virology*; D014162:Transfection
D047629:Estrogen Receptor beta; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins