Protein Engineering, Vol. 12, No. 12, 1035-1040,
December 1999
© 1999 Oxford University Press
COMMUNICATIONS |
Attenuation of green fluorescent protein half-life in mammalian cells
CRC Chromosome Molecular Biology Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of ~26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives. A C-terminal fusion of a PEST sequence from the mouse ornithine decarboxylase gene reduced the half-life to 9.8 h, resulting in a GFP variant suitable for the study of dynamic cellular processes. In an N-terminal fusion containing the mouse cyclin B1 destruction box, it was reduced to 5.8 h, with most degradation taking place at metaphase. The combination of both sequences produced a similar GFP half-life of 5.5 h. Thus, the stability of this marker protein can be controlled in predetermined ways by addition of the appropriate proteolytic signals.
Keywords: cyclin destruction box/green fluorescent protein/half-life/PEST sequence
1 To whom correspondence should be addressed. E-mail: chris{at}bioch.ox.ac.uk
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Simonen, Y. Ibig-Rehm, G. Hofmann, J. Zimmermann, G. Albrecht, M. Magnier, V. Heidinger, and D. Gabriel High-Content Assay to Study Protein Prenylation J Biomol Screen, July 1, 2008; 13(6): 456 - 467. [Abstract] [PDF]
|
|
|
|
 |
|
 T. Sunabori, A. Tokunaga, T. Nagai, K. Sawamoto, M. Okabe, A. Miyawaki, Y. Matsuzaki, T. Miyata, and H. Okano Cell-cycle-specific nestin expression coordinates with morphological changes in embryonic cortical neural progenitors J. Cell Sci., April 15, 2008; 121(8): 1204 - 1212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. V. Barysheva, J. Fujii, G. Takaesu, and S.-i. Yoshida Application of unstable Gfp variants to the kinetic study of Legionella pneumophila icm gene expression during infection Microbiology, April 1, 2008; 154(4): 1015 - 1025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Galla, A. Schambach, G. J. Towers, and C. Baum Cellular Restriction of Retrovirus Particle-Mediated mRNA Transfer J. Virol., March 15, 2008; 82(6): 3069 - 3077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Davis, E. S. Ng, M. Costa, A. K. Mossman, K. Sourris, A. G. Elefanty, and E. G. Stanley Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors Blood, February 15, 2008; 111(4): 1876 - 1884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chauvin and O. Jean-Jean Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation RNA, February 1, 2008; 14(2): 240 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Xia, M. Ayala, B. R. Thiede, and S.-C. Zhang In Vitro- and In Vivo-Induced Transgene Expression in Human Embryonic Stem Cells and Derivatives Stem Cells, February 1, 2008; 26(2): 525 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. K. Nun, D. J. Kroll, N. H. Oberlies, D. D. Soejarto, R. J. Case, P. Piskaut, T. Matainaho, C. Hilscher, L. Wang, D. P. Dittmer, et al. Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma associated herpesvirus Mol. Cancer Ther., August 1, 2007; 6(8): 2360 - 2370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. McKean, J. K. Davies, and R. J. Moore Expression of phospholipase D, the major virulence factor of Corynebacterium pseudotuberculosis, is regulated by multiple environmental factors and plays a role in macrophage death Microbiology, July 1, 2007; 153(7): 2203 - 2211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Weber, J. Stelling, M. Rimann, B. Keller, M. Daoud-El Baba, C. C. Weber, D. Aubel, and M. Fussenegger A synthetic time-delay circuit in mammalian cells and mice PNAS, February 20, 2007; 104(8): 2643 - 2648. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bartoli, N. Bourg, D. Stockholm, F. Raynaud, A. Delevacque, Y. Han, P. Borel, K. Seddik, N. Armande, and I. Richard A Mouse Model for Monitoring Calpain Activity under Physiological and Pathological Conditions J. Biol. Chem., December 22, 2006; 281(51): 39672 - 39680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Wagner and W. F. Marzluff ZFP100, a Component of the Active U7 snRNP Limiting for Histone Pre-mRNA Processing, Is Required for Entry into S Phase. Mol. Cell. Biol., September 1, 2006; 26(17): 6702 - 6712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Suzuki, O. Ohneda, N. Minegishi, M. Nishikawa, T. Ohta, S. Takahashi, J. D. Engel, and M. Yamamoto Combinatorial Gata2 and Sca1 expression defines hematopoietic stem cells in the bone marrow niche PNAS, February 14, 2006; 103(7): 2202 - 2207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Knaan-Shanzer, M. J.M. van de Watering, I. van der Velde, M. A.F.V. Goncalves, D. Valerio, and A. A.F. de Vries Endowing Human Adenovirus Serotype 5 Vectors with Fiber Domains of Species B Greatly Enhances Gene Transfer into Human Mesenchymal Stem Cells Stem Cells, October 1, 2005; 23(10): 1598 - 1607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Yoshizaki, Y. Tago, Y. Takeuchi, E. Sawatari, T. Kobayashi, and T. Takeuchi Green Fluorescent Protein Labeling of Primordial Germ Cells Using a Nontransgenic Method and Its Application for Germ Cell Transplantation in Salmonidae Biol Reprod, July 1, 2005; 73(1): 88 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Ng, L. Azzola, K. Sourris, L. Robb, E. G. Stanley, and A. G. Elefanty The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells Development, March 1, 2005; 132(5): 873 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Voon, L. S. Subrata, S. Baltic, M. P. Leu, J. M. Whiteway, A. Wong, S. A. Knight, F. T. Christiansen, and J. M. Daly Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system Nucleic Acids Res., February 16, 2005; 33(3): e27 - e27. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. G. Schoch, A. Lori, K. A. Burns, T. Eldred, J. C. Olsen, and S. H. Randell A subset of mouse tracheal epithelial basal cells generates large colonies in vitro Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L631 - L642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Gandara, P. Lopez, R. Hernando, J. G. Castano, and S. Alemany The COOH-Terminal Domain of Wild-Type Cot Regulates Its Stability and Kinase Specific Activity Mol. Cell. Biol., October 15, 2003; 23(20): 7377 - 7390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Fortin, J. Bouthillier, and F. Marceau High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1647 - H1654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Houle, G. Molinaro, A. Adam, and F. Marceau Tissue Kallikrein Actions at the Rabbit Natural or Recombinant Kinin B2 Receptors Hypertension, March 1, 2003; 41(3): 611 - 617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Nolan, T. R. Sarafi-Reinach, J. G. Horne, A. M. Saffer, and P. Sengupta The DAF-7 TGF-beta signaling pathway regulates chemosensory receptor gene expression in C. elegans Genes & Dev., December 1, 2002; 16(23): 3061 - 3073. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Vadolas, H. Wardan, M. Orford, L. Voullaire, F. Zaibak, R. Williamson, and P. A. Ioannou Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human beta -globin locus in erythropoietic cells Blood, December 1, 2002; 100(12): 4209 - 4216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Heremans, M. Van De Casteele, P. in't Veld, G. Gradwohl, P. Serup, O. Madsen, D. Pipeleers, and H. Heimberg Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3 J. Cell Biol., October 28, 2002; 159(2): 303 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Kamau, F. Grimm, and A. B. Hehl Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro Antimicrob. Agents Chemother., December 1, 2001; 45(12): 3654 - 3656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Bettinger, R. C. Carlisle, M. L. Read, M. Ogris, and L. W. Seymour Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells Nucleic Acids Res., September 15, 2001; 29(18): 3882 - 3891. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liu, J. A. Schrager, G. D. Lange, and J. W. Marsh HIV Nef-mediated Cellular Phenotypes Are Differentially Expressed as a Function of Intracellular NEF Concentrations J. Biol. Chem., August 24, 2001; 276(35): 32763 - 32770. [Abstract] [Full Text] [PDF]
|
|