Abstract
UBE1 plays an important role in the first step of ubiquitin–proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB–UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Weekes J, Morrison K, Mullen A et al (2003) Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics 3(2):208–216. doi:10.1002/pmic.200390029
Willis MS, Patterson C (2006) Into the heart: the emerging role of the ubiquitin–proteasome system. J Mol Cell Cardiol 41:567–579. doi:10.1016/j.yjmcc.2006.07.015
Hicke L (1997) Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins. FASEB J 11:1215–1226
Passmore LA, Barford D (2004) Getting into position: the catalytic mechanisms of protein ubiquitylation. Biochem J 379:513–525. doi:10.1042/BJ20040198
Yeh ETH, Gong LM, Kamitani T (2000) Ubiquitin-like proteins: new wines in new bottles. Gene 248:1–14. doi:10.1016/S0378-1119(00)00139-6
Pelzer C, Kassner I, Matentzoglu K et al (2007) UBE1L2, a novel E1 enzyme specific for ubiquitin. J Biol Chem 282(32):23010–23014. doi:10.1074/jbc.C700111200
Jin J, Li X, Gygi SP et al (2007) Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 447(7148):1135–1138. doi:10.1038/nature05902
Dou T, Gu S, Liu J et al (2005) Isolation and characterization of ubiquitin-activating enzyme E1-domain containing 1, UBE1DC1. Mol Biol Rep 32(4):265–271. doi:10.1007/s11033-005-4822-y
Stephen AG, Trausch-Azar JS, Handley-Gearhart PM et al (1997) Identification of a Region within the Ubiquitin-activating Enzyme Required for Nuclear Targeting and Phosphorylation. J Biol Chem 272(16):10895–10903. doi:10.1074/jbc.272.16.10895
Cook JC, Chock PB (1992) Isoforms of mammalian ubiquitin-activating enzyme. J Biol Chem 267:24315–24321
Schwartz AL, Trausch JS, Ciechanover A et al (1992) Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in Hepg2 cells. Proc Natl Acad Sci USA 89:5542–5546. doi:10.1073/pnas.89.12.5542
Trausch JS, Grenfell SJ, Handley-Gearhart PM et al (1993) Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton. Am J Physiol 264:C93–C102
Haas AL, Siepmann TJ (1997) Pathways of ubiquitin conjugation. FASEB J 11:1257–1268
Handley-Gearhart PM, Stephen AG, Trausch-Azar JS et al (1994) Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs. J Biol Chem 269:33171–33178
Stephen AG, Trausch-Azar JS, Ciechanover A et al (1996) The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cell cycle-dependent manner. J Biol Chem 271:15608–15614. doi:10.1074/jbc.271.26.15608
Glickman MH, Ciechanover A (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol 82:373–428
Hatfield PM, Vierstra RD (1992) Multiple forms of ubiquitin-activating enzyme El from wheat. J Biol Chem 267(21):14799–14803
Hatfield PM, Callis J, Vierstra RD (1990) Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli. J Biol Chem 265(26):15813–15817
Hatfield PM, Vierstra RD (1989) Ubiquitin-dependent proteolytic pathway in wheat germ: isolation of multiple forms of ubiquitin-activating enzyme, El. Biochemistry 28:735–742. doi:10.1021/bi00428a048
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgran quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3
Handley PM, Mueckler M, Siegel NR et al (1991) Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1. Proc Natl Acad Sci USA 88(1):258–262. doi:10.1073/pnas.88.1.258
Burger AM, Seth AK (2004) The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications. Eur J Cancer 40:2217–2229. doi:10.1016/j.ejca.2004.07.006
Hershko A (1999) Mechanisms and regulation of the degradation of cyclin B. Philos Trans R Soc Lond B Biol Sci 354:1571–1576. doi:10.1098/rstb.1999.0500
Szczepanowski RH, Filipek R, Bochtler M (2005) Crystal structure of a fragment of mouse ubiquitin-activating enzyme. J Biol Chem 280:22006–22011. doi:10.1074/jbc.M502583200
Lee I, Schindelin H (2008) Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell 134:268–278. doi:10.1016/j.cell.2008.05.046
Acknowledgments
This work is supported by National Basic Research Program of China (973 Program, 2007CB914304) and National Natural Science Foundation of China (30770427) and New Century Excellent Talents in University (NCET-06-0356) and Shanghai Leading Academic Discipline Project (B111) and National Talent Training Fund in Basic Research of China (No. J0630643).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zheng, M., Liu, J., Yang, Z. et al. Expression, purification and characterization of human ubiquitin-activating enzyme, UBE1. Mol Biol Rep 37, 1413–1419 (2010). https://doi.org/10.1007/s11033-009-9525-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-009-9525-3