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

Protein Expression and Purification
Peili ChenYusen Liu

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 yie...Continue Reading

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Citations

Mar 31, 2010·Infection and Immunity·Timothy T CornellThomas P Shanley
Mar 31, 2012·American Journal of Physiology. Gastrointestinal and Liver Physiology·Ranyia MattaYusen Liu
Jun 12, 2012·American Journal of Physiology. Lung Cellular and Molecular Physiology·Timothy T CornellThomas P Shanley
Mar 29, 2014·Nature Communications·Ainhoa Sánchez de DiegoKarel H M van Wely
Mar 28, 2008·Proceedings of the National Academy of Sciences of the United States of America·Xiaoming ZhouMaurice B Burg
Sep 9, 2004·Proceedings of the National Academy of Sciences of the United States of America·Kei OhnumaChikao Morimoto
Jan 15, 2016·Biochemistry·Michael WeyJongyun Heo
Oct 16, 2004·The Journal of Biological Chemistry·Edward G ShepherdYusen Liu
Sep 14, 2018·Science Translational Medicine·Frederique M F CornelisRik J Lories
May 31, 2007·The Journal of Biological Chemistry·Traci R LyonsC Kenneth Kassenbrock
Nov 16, 2006·Journal of Cell Science·Katarina HattulaJohan Peränen
May 29, 2007·Methods : a Companion to Methods in Enzymology·Andreas Vogt, John S Lazo

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