A mammalian expression system for rapid production and purification of active MAP kinase phosphatases.
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.
Chen
Peili
P
Stress Signaling Unit, Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Hutter
Dorothy
D
Liu
Pinghu
P
Liu
Yusen
Y
D000818:Animals; D018797:Cell Cycle Proteins; D054638:Dual Specificity Phosphatase 1; D054637:Dual-Specificity Phosphatases; D005822:Genetic Vectors; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D054639:Mitogen-Activated Protein Kinase Phosphatases; D051059:PTEN Phosphohydrolase; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D025521:Tumor Suppressor Proteins; D054639:Mitogen-Activated Protein Kinase Phosphatases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases; C517019:DUSP1 protein, human; C490583:DUSP4 protein, human; D054638:Dual Specificity Phosphatase 1; D054637:Dual-Specificity Phosphatases; C517021:Dusp1 protein, rat; C094132:Dusp4 protein, rat; D017027:Protein Tyrosine Phosphatases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human