title: Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli.
abstract: Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Unit of Microbiology, Uppsala University, Box 582, S-751 23 Uppsala, Sweden.
authors.forename: B G
authors.initials: BG
authors.lastname: Yue
authors.forename: P
authors.initials: P
authors.lastname: Ajuh
authors.forename: G
authors.initials: G
authors.lastname: Akusjärvi
authors.forename: A I
authors.initials: AI
authors.lastname: Lamond
authors.forename: J P
authors.initials: JP
authors.lastname: Kreivi
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D012995:Solubility
chemical_list: D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000068103:Serine-Arginine Splicing Factors; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases