TY - JOUR
T1 - Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis
AU - Acharya, Asha
AU - Xu, Xin Jing
AU - Husain-Ponnampalam, Rhonda D.
AU - Hoffmann-Benning, Susanne
AU - Kuo, Min Hao
N1 - Funding Information:
Authors thank Steven J. Triezenberg and R. William Henry for advice and Kuo lab members for helpful discussions, Shelley Berger for the GST-p53 construct, and Anastasia Gridasova and Marc Anderson for help with plasmid construction. This work was supported by NIH (R01 GM62282) and NSF (MCB 0315542) grants to M.-H.K. and a post-doctoral fellowship from MSU Foundation to A.A.
PY - 2005/6
Y1 - 2005/6
N2 - 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.
AB - 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.
KW - Acetylation
KW - Tethered catalysis
KW - Yeast two-hybrid system
KW - p53
UR - http://www.scopus.com/inward/record.url?scp=18244369794&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18244369794&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2005.01.015
DO - 10.1016/j.pep.2005.01.015
M3 - Article
C2 - 15866730
AN - SCOPUS:18244369794
SN - 1046-5928
VL - 41
SP - 417
EP - 425
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -