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Expression, purification, and characterization of a recombinant flavin reductase from the luminescent marine bacterium Photobacterium leiognathi
Author(s) -
Crowley Thomas E.
Publication year - 2010
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.20348
Subject(s) - flavin mononucleotide , flavin group , chemistry , photobacterium , biochemistry , affinity chromatography , luciferase , chromatography , enzyme , bacteria , biology , vibrio , gene , transfection , genetics
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 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 340 as the redox partner, NADH, is oxidized.