Protein Engineering, Vol. 12, No. 12, 1035-1040,
December 1999
© 1999 Oxford University Press
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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
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