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Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector

, : Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector. European Journal of Biochemistry 190(2): 291-298

The catalytic subunit of protein phosphatase 1 (PP1), a key enzyme in the regulation of many cellular functions, has been expressed in insect cells using a baculovirus vector containing PP1.alpha. cDNA. The expressed protein had the same apparent molecular mass as PP1 from rabbit skeletal muscle and comprised up to 25% of the total cellular protein. About 5% of expressed PP1.alpha. was present as a soluble active species, representing a 15-fold increase over the endogenous activity. Insoluble protein, comprising about 95% of the expressed PP1 was dissolved in 6 M guanidinium chloride and could be fully reactivated by extensive and rapid dilution with buffers containing Mn2+. By a number of criteria (specific activity towards phosphorylase, interaction with inhibitor-1, inhibitor-2 and okadaic acid), this reactivated species was indistinguishable from authentic PP1, and could be concentrated and purified to homogeneity by a single chromatography on DEAE-Sepharose. This procedure yielded about 10 mg active PP1/l culture, which will facilitate future structural analyses of native and mutant protein phosphatases.


PMID: 2163839

DOI: 10.1111/j.1432-1033.1990.tb15575.x

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