NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells.

Wadzinski BE, Eisfelder BJ, Peruski LF, Mumby MC, Johnson GL
J Biol Chem. 1992 267 (24): 16883-8

PMID: 1380955

Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.

MeSH Terms (19)

Amino Acid Sequence Animals Antibodies, Monoclonal Base Sequence Cell Line DNA, Viral Epitopes Hemagglutinin Glycoproteins, Influenza Virus Hemagglutinins, Viral Kinetics Macromolecular Substances Molecular Sequence Data Oligodeoxyribonucleotides Phosphoprotein Phosphatases Polymerase Chain Reaction Protein Phosphatase 2 Recombinant Proteins Restriction Mapping Viral Envelope Proteins

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