A mammalian expression system for rapid production and purification of active MAP kinase phosphatases.

Abstract	Expression of enzymatically active mammalian proteins in Escherichia coli can proven to be a challenging task due to poor solubility, improper folding, and lack of adequate posttranslational modification. Expression of mammalian proteins using baculovirus or yeast systems is time-consuming and may also be subject to inadequate modification. In order to overcome these technical difficulties, we have developed a mammalian expression system for the convenient subcloning of cDNA fragments, high-level expression, and one-step purification of enzymatically active proteins. The mammalian expression vector pEBG that expresses glutathione S-transferase fusion proteins was modified to create an SrfI restriction site in the multiple cloning site. The protein coding sequences of MAP kinase phosphatase-1 (MKP-1), MAP kinase phosphatase-2 (MKP-2), and the tumor suppressor PTEN were PCR-amplified using Pfu DNA polymerase and cloned into the SrfI site through SrfI digestion-coupled ligation. The resulting plasmids were transiently transfected into 293T cells using FuGENE 6 transfection reagent. Forty eight hours after transfection, cells were harvested and bioactive recombinant proteins were purified by glutathione-Sepharose beads. Protein yield, which ranged from 200 to 700 microg, was more than adequate for biochemical studies. The usefulness of this versatile system for studying protein function and its potential application for proteomics research are discussed.
Authors	Peili Chen, Dorothy Hutter, Pinghu Liu, Yusen Liu
Journal	Protein expression and purification (Protein Expr Purif) Vol. 24 Issue 3 Pg. 481-8 (Apr 2002) ISSN: 1046-5928 [Print] United States
PMID	11922765 (Publication Type: Journal Article)
Copyright	Copyright 2002 Elsevier Science (USA).
Chemical References	Cell Cycle Proteins Immediate-Early Proteins Tumor Suppressor Proteins Mitogen-Activated Protein Kinase Phosphatases Phosphoprotein Phosphatases Protein Phosphatase 1 Protein Phosphatase 2 Phosphoric Monoester Hydrolases DUSP1 protein, human DUSP4 protein, human Dual Specificity Phosphatase 1 Dual-Specificity Phosphatases Dusp1 protein, rat Dusp4 protein, rat Protein Tyrosine Phosphatases PTEN Phosphohydrolase PTEN protein, human
Topics	Animals Cell Cycle Proteins Dual Specificity Phosphatase 1 Dual-Specificity Phosphatases Genetic Vectors Humans Immediate-Early Proteins (genetics, isolation & purification, metabolism) Kinetics Mitogen-Activated Protein Kinase Phosphatases PTEN Phosphohydrolase Phosphoprotein Phosphatases Phosphoric Monoester Hydrolases (genetics, isolation & purification, metabolism) Protein Phosphatase 1 Protein Phosphatase 2 Protein Tyrosine Phosphatases (biosynthesis, genetics, isolation & purification, metabolism) Rats Tumor Suppressor Proteins (genetics, isolation & purification, metabolism)

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