Metallopanstimulin gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.
The protein product of the human Metallopanstimulin (MPS-1) gene was produced in the insect cell line Spodoptera frugiperda (Sf9) using the baculovirus expression vector system Autographa californica nuclear polyhedrosis virus (AcNPV). When a cloned MPS-1 complementary DNA sequence was inserted into the AcNPV viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately 10,000 was observed in extracts of infected Sf9 cells. This protein was not detected in Sf9 cells infected with AcNPV-MPS-1-Del, a vector in which the MPS-1 gene was deleted. The MPS-1 protein was produced at high levels in this host-vector system (congruent to 12% of total labeled soluble protein). Characterization of the MPS-1 protein extracted from Sf9 infected cells showed that it: binds zinc ions specifically; is phosphorylated; accumulates in the nucleus; is tightly bound to the nucleus; and binds to calf thymus DNA-cellulose. The MPS-1 protein constitutes one of the major proteins in the nuclear fraction of Sf9 cells and can be purified from this source to near homogeneity by a two-step procedure composed of high-performance liquid chromatography and gel electrophoresis. Antibodies were raised against selected peptide sequences of the MPS-1 protein and used to detect MPS-1 in insect cells and in various cultured mammalian cell types. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunological properties equivalent to those of MPS-1 spontaneously expressed in various human mammalian cell lines. Finally, recombinant MPS-1 generated a specific protein-DNA complex with a duplex oligomer containing a cyclic AMP-responsive element, as assessed by gel mobility shift assays. These results support the hypothesis that the MPS-1 protein may act, at least in part, by interacting with genes possessing the cyclic AMP-responsive element sequence.
Fernandez-Pol
J A
JA
Laboratory of Molecular Oncology, Department of Veterans Affairs Medical Center, St. Louis, Missouri 63106.
Klos
D J
DJ
Hamilton
P D
PD
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D015870:Gene Expression; D005822:Genetic Vectors; D008667:Metalloproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D008667:Metalloproteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins