Rapidly degrading gfp-fusion proteins and methods of use

Details Rapidly degrading gfp-fusion proteins and methods of use Rapidly degrading gfp-fusion proteins and methods of use

C - Chemistry, Metallurgy

12

N

C12N 15/62 (2006.01) C07H 21/04 (2006.01) C07K 14/00 (2006.01) C07K 14/435 (2006.01) C07K 19/00 (2006.01) C12N 1/21 (2006.01) C12N 5/10 (2006.01) C12N 9/88 (2006.01) C12N 15/11 (2006.01) C12N 15/52 (2006.01) C12N 15/63 (2006.01) C12N 15/85 (2006.01) C12N 15/86 (2006.01) G01N 33/53 (2006.01)

Patent

CA 2328888

Green fluorescent protein (GFP) is widely used as a reporter in determining gene expression and protein localization. The present invention provides fusion proteins with a half life of ten hours or less with several embodiments having half lives of 4 hours or less. Such proteins may be constructed by fusing C-terminal amino acids of the degradation domain of mouse ornithine decarboxylase (MODC), which contains a PEST sequence, to the C-terminal end of an enhanced variant of GFP (EGFP). Fluorescence intensity of the fusion protein in transfected cells is similar to that of EGFP, but the fusion protein, unlike EGFP, is unstable in the presence of cycloheximide. Specific mutations in the MODC region have resulted in mutants with varying half lives, useful for a variety of purposes.

La protéine fluorescente verte (GFP) est largement utilisée comme rapporteur pour déterminer l'expression de gènes et la localisation de protéines. La présente invention concerne des protéines de fusion ayant une demi-vie de 10 heures ou moins avec plusieurs modes de réalisation ayant des demi-vies de 4 heures ou moins. Ces protéines peuvent être construites par fusion d'acides aminés C-terminaux du domaine de dégradation de l'ornithine décarboxylase de souris (MODC), contenant une séquence PEST, à l'extrémité C-terminale d'un variant amélioré de GFP (EGFP). L'intensité de fluorescence de la protéine de fusion dans des cellules transfectées est similaire à celle d'EGFP mais la protéine de fusion, à la différence d'EGFP, est instable en la présence de cycloheximide. Des mutations spécifiques dans la région MODC ont permis d'obtenir des mutants à demi-vies variables utiles à diverses fins.

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