Expression and purification of biologically active v-sis/platelet-derived growth factor B protein by using a baculovirus vector system.
Malignant transformation induced by simian sarcoma virus is mediated by its v-sis protein, the monkey homolog of the platelet-derived growth factor (PDGF) B chain. By use of an appropriately engineered baculovirus expression vector, the v-sis protein was expressed in the insect cell line Spodoptera frugiperda (Sf9) at a level 50- to 100-fold higher than that observed with overexpression in mammalian-cell transfectants. The sis protein produced by Sf9 cells underwent processing similar to that observed in mammalian cells, including efficient disulfide-linked dimer formation. Moreover, the recombinant sis protein was capable of binding PDGF receptors and inducing DNA synthesis as efficiently as PDGF-B synthesized by mammalian cells. A significant fraction of sis protein was released from Sf9 cells, which made possible a one-step immunoaffinity purification to near homogeneity with a 40% recovery of biological activity. These results demonstrate that a protein whose normal processing requires both intrachain and interchain disulfide-bridge formation can be efficiently expressed in a biologically active form in insect cells by using a baculovirus vector system.
Giese
N
N
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20814.
May-Siroff
M
M
LaRochelle
W J
WJ
van Wyke Coelingh
K
K
Aaronson
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D020575:Oncogene Proteins v-sis; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012191:Retroviridae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D015291:Transforming Growth Factors / Q000235:genetics*
D020575:Oncogene Proteins v-sis; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; D015291:Transforming Growth Factors; D017479:Receptors, Platelet-Derived Growth Factor