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

Paper ID Volume ID Publish Year Pages File Format Full-Text
18034 42710 2007 7 PDF Available
Title
Expression, purification and immobilization of firefly luciferase on alkyl-substituted Sepharose 4B
Abstract

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.

Keywords
Firefly luciferase; Immobilization; Hydrophobic; Adsorption; Luminescence; ANS
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Expression, purification and immobilization of firefly luciferase on alkyl-substituted Sepharose 4B
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 740–746
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us