Luciferase signal enhancing compositions

Abstract

Reagents and compositions for use in reactions catalysed by luciferase enzymes, and in particular for use in luciferase-based gene reporter assays are described. The invention also provides methods and compositions for, inter alia, increasing the sensitivity and/or improving the kinetics of luciferase-catalysed reactions.

The invention claimed is: 
     
       1. A method for conducting a luciferase-based assay comprising,
 expressing within a eukaryotic cell a luciferase that is secreted in its native form, wherein the luciferase lacks a functional signal peptide such that it is expressed intracellularly in the eukaryotic cell;   recovering the activity of the luciferase by contacting the luciferase with a reagent composition that provides an environment suitable for conversion of the luciferase into its active folded form, wherein the reagent composition comprises an agent selected from the group consisting of bromide anion, chelator, redox buffer, and buffer with a pH greater than 8.0;   contacting the luciferase with a substrate of the luciferase enzyme; and   detecting or measuring the bioluminescence.   
     
     
       2. The method of  claim 1 , wherein the luciferase in its active form includes disulphide bonds between cysteine residues. 
     
     
       3. The method of  claim 1 , wherein the recovering activity comprises a cell lysis step. 
     
     
       4. The method of  claim 1 , wherein the method is employed in a reporter gene assay. 
     
     
       5. A method for conducting a luciferase-based assay comprising,
 expressing within a eukaryotic cell a luciferase that is secreted in its native form, wherein the luciferase lacks a functional signal peptide such that it is expressed intracellularly in the eukaryotic cell;   recovering the activity of the luciferase by contacting the luciferase with a reagent composition that provides an environment suitable for conversion of the luciferase into its active folded form, wherein the reagent composition comprises an oxidising agent;   contacting the luciferase with a substrate of the luciferase enzyme; and   detecting or measuring the bioluminescence.   
     
     
       6. The method of  claim 5 , wherein the luciferase in its active form includes disulphide bonds between cysteine residues. 
     
     
       7. The method of  claim 5 , wherein the recovering activity comprises a cell lysis step. 
     
     
       8. The method of  claim 5 , wherein the method is employed in a reporter gene assay. 
     
     
       9. A method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
 a) contacting the cell or sample of cells with a reagent composition comprising a detergent and one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   b) adding a substrate of the luciferase; and   c) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.   
     
     
       10. The method of claim 9, wherein step (b) occurs simultaneously with, or following step (a). 
     
     
       11. The method of claim 9, wherein the luciferase lacks a functional signal peptide. 
     
     
       12. The method of claim 9, wherein the detergent is a non-ionic or zwitterionic detergent. 
     
     
       13. A method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
 a) contacting the cell or sample of cells with a reagent composition that lyses the cell or sample of cells and provides an environment that enables conversion of the luciferase into an active state, comprising a detergent and one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   b) adding a substrate of the luciferase; and   c) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.   
     
     
       14. The method of claim 13, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state. 
     
     
       15. The method of claim 13, wherein the detergent is a non-ionic or zwitterionic detergent. 
     
     
       16. A method for increasing the bioluminescent signal generated by a recombinant luciferase, the method comprising:
 a) contacting the recombinant luciferase with a reagent composition comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   b) adding a substrate of the luciferase; and   c) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.   
     
     
       17. The method of claim 16, wherein step (b) occurs simultaneously with, or following step (a). 
     
     
       18. The method of claim 16, wherein the luciferase lacks a functional signal peptide. 
     
     
       19. The method of claim 16, wherein the reagent composition further comprises a detergent selected from a non-ionic or zwitterionic detergent. 
     
     
       20. A method for increasing the bioluminescent signal generated by a recombinant luciferase, the method comprising:
 a) contacting the recombinant luciferase with a reagent composition that provides an environment that enables conversion of the luciferase into an active state, comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   b) adding a substrate of the luciferase; and   c) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.   
     
     
       21. The method of claim 20, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state. 
     
     
       22. A method for reducing the rate of decay of the bioluminescent signal generated by a recombinant luciferase, the method comprising:
 a) contacting the recombinant luciferase with a reagent composition comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   b) adding a substrate of the luciferase; and   c) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.   
     
     
       23. The method of claim 22, wherein step (b) occurs simultaneously with, or following step (a). 
     
     
       24. The method of claim 22, wherein the luciferase lacks a functional signal peptide. 
     
     
       25. The method of claim 22, wherein the reagent composition further comprises a detergent selected from a non-ionic or zwitterionic detergent. 
     
     
       26. A method for reducing the rate of decay of the bioluminescent signal generated by a recombinant luciferase, the method comprising:
 a) contacting the recombinant luciferase with a reagent composition that provides an environment that enables conversion of the luciferase into an active state, comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   b) adding a substrate of the luciferase; and   c) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.   
     
     
       27. The method of claim 26, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.