title: Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis.
abstract: Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein, we present here a tethered catalysis approach for the production of acetylated p53 in vivo. P53 is a major tumor suppressor protein that protects the cell from various oncogenic stresses. Upon DNA damage, p53 is stabilized and activated by a plethora of post-translational modifications, including acetylation. Here, we show that constitutively acetylated p53 can be expressed and purified from both yeast and Escherichia coli. This method is highly suitable for studying protein-protein interactions in the conventional yeast two-hybrid screen that requires a constitutively acetylated state of p53. Furthermore, effective production and purification of acetylated p53 from E. coli supports future biochemical and structural characterization. The method described in this work can be applied to other proteins and modifications, and thus has widespread use in the fields of signal transduction and proteomic research.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Asha
authors.initials: A
authors.lastname: Acharya
authors.forename: Xin-Jing
authors.initials: XJ
authors.lastname: Xu
authors.forename: Rhonda D
authors.initials: RD
authors.lastname: Husain-Ponnampalam
authors.forename: Susanne
authors.initials: S
authors.lastname: Hoffmann-Benning
authors.forename: Min-Hao
authors.initials: MH
authors.lastname: Kuo
mesh_terms: D000107:Acetylation; D002384:Catalysis; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015003:Yeasts / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D008239:Lysine