Cell Growth & Differentiation, Vol 5, Issue 8 811-825, Copyright © 1994 by American Association of Cancer Research

ARTICLES

Metallopanstimulin gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA

JA Fernandez-Pol, DJ Klos and PD Hamilton
Laboratory of Molecular Oncology, Department of Veterans Affairs Medical Center, St. Louis, Missouri 63106.

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.

This article has been cited by other articles:

				Y.-w. Wang, Y. Qu, J.-f. Li, X.-h. Chen, B.-y. Liu, Q.-l. Gu, and Z.-g. Zhu In vitro and In vivo Evidence of Metallopanstimulin-1 in Gastric Cancer Progression and Tumorigenicity. Clin. Cancer Res., August 15, 2006; 12(16): 4965 - 4973. [Abstract] [Full Text] [PDF]

				J. T. Wadsworth, K. D. Somers, B. C. Stack Jr, L. Cazares, G. Malik, B.-L. Adam, G. L. Wright Jr, and O. J. Semmes Identification of Patients With Head and Neck Cancer Using Serum Protein Profiles Arch Otolaryngol Head Neck Surg, January 1, 2004; 130(1): 98 - 104. [Abstract] [Full Text] [PDF]