US 6,306,600 B1 · Rapidly degrading GFP-fusion proteins and methods of use

Abstract

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.

Claims9

Dependent claims

A method of assaying the positive or negative regulatory function of a transcriptional or translational control sequence which controls or regulates the expression of a coding sequence in a host cell with a transient fluorescent reporter protein, comprising the steps of:

a) transfecting cells with an expression vector comprising a DNA sequence that codes for a fluorescent fusion protein comprising a Green Fluorescent Protein (GFP) and a PEST sequence, wherein said fusion protein has a half life of no more than about ten hours, and wherein said expression vector contains said transcriptional or translational control sequence that may affect the expression of said fluorescent fusion protein;
b) transfecting cells with a control expression vector comprising a DNA sequence that codes for said fluorescent fusion protein, wherein said control expression vector does not contain said transcriptional or translational control sequence;
and
c) detecting a presence, absence or amount of fluorescence in said cells, wherein an increase or decrease in fluorescence in cells transfected with the vector containing the control sequence compared to cells transfected with the vector not containing the control sequence indicates a positive or negative regulatory function, respectively for said transcriptional or translational control sequence.

1 more element

Dependencies are inferred from the claim text.

Rapidly degrading GFP-fusion proteins and methods of use

Life of this patent

Rapidly degrading GFP-fusion proteins and methods of use

Life of this patent

Abstract

Claims9