A Mammalian Expression System for Rapid Production and Purification of Active MAP Kinase Phosphatases

Autor: Pinghu Liu, Peili Chen, Dorothy Hutter, Yusen Liu
Rok vydání: 2002
Předmět:
Zdroj: Protein Expression and Purification. 24:481-488
ISSN: 1046-5928
DOI: 10.1006/prep.2001.1599
Popis: 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 Srf I 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 Srf I site through Srf I 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 μg, 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.
Databáze: OpenAIRE
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