Development of a thermostable firefly luciferase

Firefly luciferase forms the basis of a wide range of analytical techniques. However, the enzyme is unstable and rapidly loses activity even at room temperature. This leads to losses in sensitivity and precision in analytical applications and also severely limits the fieldability of devices incorporating luciferase-based technologies. A number of point mutations have previously been identified that significantly increase the thermostability of the enzyme. We show here that when such mutations are combined they can have an additive effect on the stabilisation of the enzyme. As such, we have constructed a luciferase mutant containing four point mutations, relative to the wildtype enzyme, resulting in remarkably greater thermostability. © 2002 Elsevier Science B.V. All rights reserved.