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

North American firefly, Photinus pyralis, luciferase was produced in Escherichia coli BL21 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 Km 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.