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

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

J A Fernandez-Pol¹, D J Klos, P D Hamilton

¹Laboratory of Molecular Oncology, Department of Veterans Affairs Medical Center, St. Louis, Missouri 63106.

Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research|August 1, 1994

View abstract on PubMed

Summary

The human Metallopanstimulin (MPS-1) protein was successfully produced in insect cells using a baculovirus system. This recombinant MPS-1 protein binds zinc, is phosphorylated, and interacts with DNA containing cyclic AMP-responsive elements.

Area of Science:

Molecular Biology
Protein Expression
Gene Regulation

Background:

Metallopanstimulin (MPS-1) is a human gene product.
Understanding MPS-1 function requires reliable protein production for characterization.
Insect cell expression systems offer a viable method for producing eukaryotic proteins.

Purpose of the Study:

To produce and characterize the human Metallopanstimulin (MPS-1) protein in an insect cell system.
To investigate the biochemical properties and cellular localization of recombinant MPS-1.
To assess the DNA-binding activity of MPS-1, particularly its interaction with cyclic AMP-responsive elements.

Main Methods:

Baculovirus expression vector system (Autographa californica nuclear polyhedrosis virus) used to express MPS-1 in Spodoptera frugiperda (Sf9) cells.
Characterization techniques included zinc-binding assays, phosphorylation analysis, nuclear fractionation, DNA-cellulose binding, Western analysis, and gel mobility shift assays.
Antibodies were generated against MPS-1 peptides for immunological detection.

Main Results:

High-level production of a ~10,000 molecular weight MPS-1 polypeptide in infected Sf9 cells.
Recombinant MPS-1 specifically binds zinc ions, is phosphorylated, and accumulates in the nucleus.
MPS-1 tightly binds to the nucleus and calf thymus DNA-cellulose, indicating DNA-binding capability.
Western analysis confirmed the identity and properties of baculovirus-generated MPS-1 compared to mammalian cell-expressed MPS-1.
Gel mobility shift assays demonstrated that recombinant MPS-1 forms a specific complex with DNA containing a cyclic AMP-responsive element.

Conclusions:

The baculovirus expression system is effective for producing functional human MPS-1 protein.
MPS-1 exhibits properties consistent with a nuclear protein involved in gene regulation.
MPS-1's interaction with cyclic AMP-responsive elements suggests a role in regulating genes responsive to cyclic AMP signaling pathways.