Abstract: Proteins are provided having luciferase activity with greater heat stability than wildtype luciferases by replacing the glutamate equivalent to that at position 354 of Photinus pyralis luciferase or 356 of Luciola luciferases with an alternative amino acid, particularly lysine. DNA, vectors and cells that encode for and express the proteins are also provided as are test kits and reagents for carrying out luminescence assays using the proteins of the invention. Preferred proteins have a second replaced amino acid at a position equivalent to position 215 of Photinus pyralis luciferase or 217 of Luciola luciferases.