Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits α and β

Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, α and β. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit α and β isoforms, we expressed human PP2Acα and cβ in High Five insect cells. The recombinant PP2Acα and cβ possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acα or cβ was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aα/cα and Aα/cβ) with PR65α/Aα. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acα and cβ are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.