Expression of bovine activin-A and inhibin-A in recombinant baculovirus-infected Spodoptera frugiperda Sf 21 insect cells.
Currently, bioactive activin and inhibin for investigative purposes are obtained either by purification from bovine or porcine follicular fluid or have been kindly supplied in limited amounts by Genentech. The latter are recombinant formulations produced in cultured monkey kidney CV-1 cells. The aims of this study were to assess the potential of the baculovirus expression system as an alternative means to produce recombinant activin and inhibin. Towards these goals, two recombinant baculoviruses, AcBovACTA and AcBovINHA, were constructed. AcBovACTA contains a contiguous copy of the bovine beta A-inhibin/activin structural gene encoding the beta A-preproprotein whereas AcBovINHA contains contiguous copies of the bovine alpha-inhibin and beta A-inhibin/activin structural genes encoding the alpha- and beta A-preproproteins, respectively. Western blot analyses, using monoclonal antibodies specific for the mature portions of the alpha-inhibin and beta A-inhibin/activin subunits, demonstrated that Spodoptera frugiperda Sf21 cells infected with either recombinant virus secreted mature homodimeric activin-A into the medium. In addition, Sf21 cells infected with the recombinant AcBovINHA virus were found also to produce substantial amounts of the alpha-inhibin precursor protein. However, the mature portion of the latter is not secreted into the medium but is retained within infected cells in an incompletely processed form(s). The recombinant activin-A secreted by Sf21 cells infected with the AcBovACTA virus was shown to possess activin bioactivity when analysed by in vitor bioassay and, therefore, provides an alternative route to mammalian cell expression for the production of recombinant activin-A.
Cronin
C N
CN
Department of Pharmacology, University College Dublin, Belfield, Ireland.
Thompson
D A
DA
Martin
F
F
D028341:Activins; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006133:Growth Substances / Q000096:biosynthesis* / Q000235:genetics; D007265:Inhibins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000821:virology*
D006133:Growth Substances; D011994:Recombinant Proteins; D028341:Activins; D007265:Inhibins