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Phosphorylation of the eukaryotic cortactin protein by endogenous kinases in an E. coli expression system
Author(s) -
Priester Jacob,
Love Haley,
Kruchten Anne
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1038.9
Subject(s) - cortactin , phosphorylation , phosphoprotein , phosphoserine , biology , autophosphorylation , protein phosphorylation , kinase , microbiology and biotechnology , biochemistry , protein kinase a , cell , serine , cytoskeleton
Purified protein samples are often acquired via recombinant DNA techniques using heterologous E. coli induced expression systems. This bacterial expression and purification technique has been widely utilized for decades, and many researchers have worked under the misconception that eukaryotic proteins are not post‐translationally modified in a significant way during expression and purification in bacteria. Based on contradicting experimental data, we hypothesized that cortactin is phosphorylated by endogenous kinases in E. coli. We analyzed the phosphorylation state of cortactin after in vitro purification from E. coli using western blotting techniques and phosphoprotein stains. We found a heterogenous population of both phosphoserine and phosphotyrosine cortactin after protein purification. As phosphorylation of cortactin has been a significant research focus in many research labs over the last two decades, this result calls for increased scrutiny of experimental results involving in vitro phosphorylated cortactin. This work was supported by the OHSU Medical Research Foundation Award and Linfield College Faculty/Student Collaborative Research Fund.