Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector.
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 PP1/1 culture, which will facilitate future structural analyses of native and mutant protein phosphatases.
Berndt
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Department of Biochemistry, The University, Dundee, Scotland.
Cohen
P T
PT
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D007313:Insecta / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D014176:Protein Biosynthesis; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1