Luminescent markers

Abstract

The invention relates to luminescent proteins, nucleic acids encoding same, compositions and combinations comprising the proteins, and methods using the proteins, nucleic acids, compositions and combinations. In particular, a luminescent protein is provided comprising oncomodulin in which a salt bridge has been introduced to provide greater stability. The protein may be used as a luminescent marker in, for example, luminescent items, immunoassays, and fluorescent energy transfer assays.

1 . A luminescent protein comprising oncomodulin in which one or more salt bridge and/or hydrogen bonding network has been introduced to provide greater stability when compared to a native oncomodulin or CDOM33. 
     
     
         2 . A luminescent protein comprising oncomodulin in which a CD-loop of oncomodulin is replaced with a more potent metal binding site and Phe47 is replaced with Trp47. 
     
     
         3 . A luminescent protein as claimed in  claim 1  further comprising one or more of the following: (a) Gln27 of oncomodulin is replaced with Lys27, (b) Ser36 of oncomodulin is replaced with Lys36, (c) an extended C-terminus, (d) enhanced Tb3+ luminescence relative to CDOM33, (e) has long-lived luminescence, and (f) a large Stoke's shift with long wavelength emission. 
     
     
         4 . A luminescent protein as claimed in  claim 2  wherein the CD loop of oncomodulin is replaced with DKNADGWIEFEE (SEQ ID NO. 13). 
     
     
         5 . A luminescent protein as claimed  claim 1  comprising an amino acid sequence of SEQ ID NO. 5. 
     
     
         6 . A truncation, an analog, or a protein having substantial sequence identity to a protein as claimed in  claim 1 . 
     
     
         7 . A chimeric protein comprising a protein as claimed in  claim 1  associated or conjugated with one or more molecule. 
     
     
         8 . A chimeric protein as claimed in  claim 7  wherein the molecule is a luminescent agent acceptor or target peptide. 
     
     
         9 . A chimeric protein as claimed in  claim 8  wherein the luminescent agent acceptor is a green fluorescent protein. 
     
     
         10 . A chimeric protein as claimed in  claim 1  comprising (a) a luminescent protein as claimed in  claim 1  and a green fluorescent protein (GFP), (b) a luminescent protein as claimed in any preceding claim and an enzyme recognition site, or (c) a luminescent protein as claimed in any preceding claim, GFP, and an enzyme recognition site. 
     
     
         11 . A chimeric protein comprising a sequence of SEQ ID NO. 6 or SEQ ID NO. 8. 
     
     
         12 . A chimeric protein of  claim 10  (b) or (c) wherein the enzyme recognition site is a protease recognition site. 
     
     
         13 . An isolated polynucleotide encoding a luminescent protein or a chimeric protein as claimed in  claim 1 . 
     
     
         14 . An isolated polynucleotide as claimed in  claim 13  that additionally encodes a component of a bioluminescence generating system. 
     
     
         15 . An isolated polynucleotide as claimed in  claim 13  wherein the chimeric protein comprises a luminescent agent acceptor, preferably green fluorescent protein (GFP), more preferably EGFP encoded by the sequence of SEQ ID NO.2 or comprising the sequence of SEQ ID NO 7. 
     
     
         16 . A polynucleotide comprising a sequence of nucleotides of SEQ ID NO.1 or 3, or that hybridizes under moderate or high stringency to the sequence of nucleotides of SEQ ID NO. 1 or 3. 
     
     
         17 . A vector comprising a polynucleotide as claimed in  claim 1 . 
     
     
         18 . A host cell comprising a vector as claimed in  claim 1 . 
     
     
         19 . A method for preparing a luminescent protein comprising (a) transferring a vector as claimed in  claim 1  into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein. 
     
     
         20 . A protein as claimed in  claim 1  in combination with at least one component of a bioluminescence-generating system. 
     
     
         21 . A protein as claimed in  claim 20  wherein the bioluminescence generating system is a green fluorescent protein system. 
     
     
         22 . An antibody that specifically binds to a protein or chimeric protein as claimed in  claim 1 . 
     
     
         23 . A composition comprising a protein or chimeric protein as claimed in  claim 1 . 
     
     
         24 . A composition as claimed in  claim 23  wherein the protein or chimeric protein are formulated for use in luminescent items, immunoassays, fluorescent energy transfer assays, fluorescent resonance energy transfer assays, HTRF homogeneous time-resolved fluorescence assays, or in conjunction with multi-well assay devices containing integrated photo detectors. 
     
     
         25 . A combination comprising a first composition containing a protein or chimeric protein as claimed in  claim 1  or mixtures thereof and a second composition containing one or more components of a bioluminescence-generating system. 
     
     
         26 . A complex comprising a luminescent protein of  claim 1  with a metal ion. 
     
     
         27 . A complex as claimed in  claim 26  wherein the metal ion is a lanthanide ion or analog or derivative thereof, preferably a terbium ion. 
     
     
         28 . A method of using a complex as claimed in  claim 1  in energy transfer between the complex and a luminescent agent acceptor. 
     
     
         29 . A method of detecting energy transfer in a sample comprising: (a) contacting the sample with a complex as claimed in  claim 1  and a luminescent agent acceptor; (b) illuminating the sample at a wavelength suitable for excitation of the complex; and (c) measuring the amount of luminescence energy transfer from the complex to the luminescent agent acceptor 
     
     
         30 . A method of measuring the distance between a first atom and second atom or assaying the interaction between the first atom and second atom comprising: (a) coupling a complex as claimed in  claim 1  to the first atom and a luminescent agent acceptor to the second atom;
 and (b) measuring the distance between the first and second atoms, or assaying the interaction of the first and second atoms. 
 
     
     
         31 . A method as claimed in  claim 30  wherein the distance is measured by detecting energy transfer between the complex and the acceptor. 
     
     
         32 . A method for assaying interactions between a first and second molecule comprising: (a) contacting, in the presence of a lanthanide, a first molecule labelled with a luminescent protein as claimed in  claim 1  and a second molecule labelled with a luminscent agent acceptor to provide a reaction mixture; (b) exposing the reaction mixture to light at a first wavelength capable of exciting lanthanide bound to the luminescent protein and transferring energy to the luminescent agent acceptor; (c) detecting luminescense of the first molecule, second molecule, and/or lanthanide at selected wavelengths, wherein detection of luminescense of the second molecule indicates an interaction between the first molecule and second molecule. 
     
     
         33 . A method as claimed in  claim 32  which further comprises in step (a) adding a test substance which potentially may modulate the interaction. 
     
     
         34 . A method of detecting dissociation of a species comprising: (a) providing a species that is labelled with a complex as claimed in  claim 1  and a luminescent agent acceptor such that luminescence energy transfer occurs between the protein in the complex and the acceptor;
 (b) exposing the species to a condition that is capable of dissociating the species such that the complex and acceptor are no longer capable of luminescence energy transfer; (c) illuminating the species at a wavelength suitable for excitation of the complex; (d) measuring an amount of luminescence energy transfer from the complex to the acceptor; and (e) correlating the amount of luminescence energy transfer from the complex to the acceptor with the dissociation of the species 
 
     
     
         35 . A method of using a protein as claimed in  claim 1  as a detectable label. 
     
     
         36 . A method for detecting analytes in a sample comprising: (a) coupling a luminescent protein as claimed in  claim 1  to a reagent capable of selectively binding the analytes; (b) contacting the sample with the protein coupled to the reagent; (c) exposing the sample to light of an appropriate wavelength that excites the protein; and (d) detecting luminescence of the protein thereby detecting analytes in the sample. 
     
     
         37 . A method of using a protein, composition, or complex as claimed in  claim 1  to provide fluorescent illumination of novelty items or to detect and visualize infectious agents. 
     
     
         38 . A method of using a protein, complex, or composition as claimed in  claim 1  in an immunoassay or an in vitro fluorescent-based screening assay. 
     
     
         39 . A method for diagnosis and visualization of tissues in vivo or in situ using compositions containing a luminescent protein, chimeric protein, or complex as claimed in  claim 1 . 
     
     
         40 . A kit comprising a luminescent protein, chimeric protein, or complex as claimed in  claim 1 . 
     
     
         41 . A kit comprising a first composition containing a luminescent protein as claimed in  claim 1  formulated for systemic administration, local, or topical application; and a second composition containing one or more components of a bioluminescence generating system, formulated for systemic, topical or local administration; and instructions for administration.