Open AccessRenaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector
Author(s) -
BERNDT Norbert,
COHEN Patricia T. W.
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb15575.x
Subject(s) - phosphatase , protein phosphatase 1 , okadaic acid , biochemistry , complementary dna , biology , enzyme , protein subunit , microbiology and biotechnology , chemistry , gene
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 PP1α 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 PP1α 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 Mn 2+ . 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.