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HighlightsThe recombinant human PP2A catalytic subunit (rhPP2Ac) was produced in the baculovirus expression system with High Five insect cells.The expression at 19 °C can produce the rhPP2Ac with a higher activity and in a larger quantity than in the incubation conducted at 27 °C.To examine the effects of the low temperature expression on other phosphatases, we expressed human PP2B and PP2C in High Five insect cells.Optimizing the expression temperature in a baculovirus system is effective for producing a recombinant protein.