Expression, purification and immobilization of firefly luciferase on alkyl-substituted Sepharose 4B

Abstract

North American firefly, Photinus pyralis, luciferase was produced in Escherichia coli BL 21 by using a pET expression vector. The His-tagged luciferase was purified by metal-ion affinity chromatography. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. Alkyl-substituted Sepharose 4B was used for luciferase immobilization. Matrices were prepared by the glycidyl ether method with different degrees of substitution and alkyl chain length, have been used as a non-ionic matrix for immobilization of firefly luciferase through hydrophobic interaction. Exposure of hydrophobic clusters in the protein molecule was confirmed by fluorescence studies using 8-anilino-1-naphthalene-sulfonate as a hydrophobic reporter probe. Immobilization of firefly luciferase took place with relatively retention of their basic kinetic properties such as K m and recovery of activity. The simplicity of adsorption of luciferase on these matrices together with the activity reusability suggests that the method of immobilization was described provide a useful procedure of bioassays when coupled to the firefly luminescence reaction.