Multiplex assays with multiple luciferases reporters and uses thereof

摘要

The present invention encompasses modified luciferases, methods for making modified luciferases, and assays utilizing modified luciferases. Modified luciferases of the invention show increased activity over wildtype luciferases and also show increased stability of signal. The present invention also encompasses multiplex assays utilizing multiple luciferases reporters with different emission spectra and different substrates for simultaneous luciferase measurements.

主权项

1. A multiplexed luciferase assay composition comprising:
multiple luciferase reporters, wherein at least two of the luciferase reporters are selected from the group consisting of a firefly luciferase, a Renilla luciferase, a Gaussia luciferase and a Cypridina (Vargula) luciferase, and wherein the different luciferase reporters emit at different wavelengths and/or utilize different substrates, and wherein the firefly luciferase is a red-emitting human codon optimized luciferase encoded by SEQ ID NO:3 with an emission maximum of approximately 617 nm; or wherein the firefly luciferase is a green-emitting human codon optimized luciferase encoded by SEQ ID NO:4 with an emission maximum of approximately 550 nm; or wherein the Renilla luciferase comprises A55T, S130A, K136R, A143M, M185V, M253L, and S287L mutations or AI23S, D154M, E155G, D162E, I163L, and V185L mutations compared to wildtype Renilla luciferase; or wherein the Vargula luciferase is encoded by SEQ ID NO: 6; and wherein at least one of the luciferase reporters comprises a secretory signal, wherein the secretory signal is peptide sequence of SEQ ID NO: 7.