Bioimage A/s

Novo Nordisk A/s

Sara Petersen Bj&oslash;rn

Ole Thastrup

Lars Kongsbak Poulsen

S&oslash;ren Tullin

Kurt Scudder

Soren Tullin

Kasper Almholt

The present invention relates to novel variants of the fluorescent protein GFP having improved fluorescence properties.

Fluorescent proteins

The present invention relates to a method of detecting biologically active substances affecting intracellular processes, an isolated DNA, a vector and a cell for use in the method. More specifically, the present invention provides a method for detecting a biologically active substance that affects intracellular processes mediated through a protein kinase or a second messenger, which comprises incubating a cell with a test substance and measuring any change caused by the test substance in the flourescence of (i) a wild-type or modified green flourescent protein (GFP) having a protein kinase recognition site, (ii) a modified GFP containing a second messenger binding domain, or (iii) a hybrid polypeptide having a wild-type or modified GFP and an attached protein kinase recognition site or a second messenger binding domain.

Method of detecting biologically active substances

Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Flourescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tryosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnkl, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signaling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of influence. Measurement of redistribution is performed by recording of light intensity, flourescence lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a flourescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating a disease related to the function of the component.

Method for extracting quantitative information relating to an influence on a cellular response

Growing community of inventors

Lyngby, Denmark

Sara Petersen Bjørn

Growing community of inventors

Lyngby, Denmark

Sara Petersen Bj&oslash;rn

Sara Petersen Bjørn