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ERIC Number: EJ895537
Record Type: Journal
Publication Date: 2010
Pages: 10
Abstractor: As Provided
Reference Count: 26
ISSN: ISSN-1470-8175
Expression, Purification, and Characterization of a Recombinant Flavin Reductase from the Luminescent Marine Bacterium "Photobacterium Leiognathi": A Set of Exercises for Students
Crowley, Thomas E.
Biochemistry and Molecular Biology Education, v38 n3 p151-160 May-Jun 2010
In "Photobacterium," the flavin reductase encoded by "lux"G regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a light-emitting reaction. A set of experiments, that employs a "lux"G-expression plasmid construct (pGhis) and is suitable for an undergraduate laboratory course, is presented. Hexahistidine-tagged protein is expressed in "E. coli" from pGhis, with the T7 RNA polymerase/"lac" repressor induction system. Bacteria are lysed by sonication and the tag allows for purification by immobilized metal ion affinity chromatography. A gel filtration column is used to remove ions and the other small molecules. The Bradford assay, with multiwell plates and an automated plate reader, is used to identify protein concentration peaks from both columns. The concentration of purified enzyme is then calculated from its A[subscript 280] using the predicted extinction coefficient. Yield and purity are further assayed with SDS-PAGE. Activity of purified enzyme is measured with riboflavin or FMN as substrate. Reaction rate is quantified by monitoring decrease in A[subscript 340] as the redox partner, NADH, is oxidized. (Contains 7 figures and 6 tables.)
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Publication Type: Journal Articles; Reports - Descriptive
Education Level: Higher Education; Postsecondary Education
Audience: Students; Teachers
Language: English
Sponsor: N/A
Authoring Institution: N/A