Phosphorylation of glutathione-S-transferase by protein kinase C-alpha implications for affinity-tag purification.
Expression and purification of proteins as fusions with glutathione S-transferase (GST) is a standard and widely employed system. In more than 2,500 published studies, GST has been used to facilitate the purification of recombinant proteins, assess protein-protein interactions, and establish protein function. In this report, we provide evidence that GST can be phosphorylated in vitro by protein kinase C-alpha (PKC-alpha) at Ser-93. Therefore, since GST itself may be a target for a number of catalytic enzymes, failure to remove the GST tag from the recombinant protein may lead to inaccurate conclusions.
Rodriguez
Patricia
P
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, USA. rodrigpa@email.uc.edu
Mitton
Bryan
B
Kranias
Evangelia G
EG
D000818:Animals; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011993:Recombinant Fusion Proteins; D012694:Serine; D005982:Glutathione Transferase; D051571:Protein Kinase C-alpha