Expression, purification, and characterization of a recombinant flavin reductase from the luminescent marine bacterium Photobacterium leiognathi

Abstract

In Photobacterium, the flavin reductase encoded by luxG regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a lightemitting reaction. A set of experiments, that employs a luxG-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.Keywords: recombinant protein expression, immobilized metal ion affinity chromatography, LuxG, flavin reductase.Bioluminescence occurs in a variety of organisms; e.g., firefly, marine invertebrates and bacteria. Most of the luminescent bacteria exist in symbiosis with fish, proliferating within certain tissues and making the fish appear to be luminescent. The reaction that generates the light energy is catalyzed by the luciferase enzyme in all these organisms. However, the substrates of the reaction vary quite a bit. In the luminescent marine bacteria Photobacterium leiognathi and Photobacterium fischeri (previously known as Vibrio fischeri), one of the substrates is the fully-reduced, quinone form of flavin mononucleotide (FMN):As the light-generating reaction involves oxidation of FMNH 2 to FMN, sustained luminescence necessitates the recycling of FMN back to the reduced form. Such a reaction (Fig. 1) is usually catalyzed by a flavin reductase enzyme [2] (the reduced form is often shown as FMNH 2 , however at pH > 6.3 it ionizes to FMNH 2 [3]). NADH is oxidized as FMN is reduced.In P. fischeri and P. leiognathi the luciferase enzyme is an ab heterodimer [4]. The genes for these subunits, luxA and luxB, are encoded within the lux operon [5][6][7][8]. This operon also includes luxG that codes for a flavin reductase (protein is designated LuxG [9]). Thus, the translation of the lux operon-encoded mRNA should generate equimolar amounts of luciferase enzyme and flavin reductase.

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection

“…Flavin reductase activity was measured by monitoring the decrease of absorbance at 340 nm as NADH was depleted using FMN as the electron acceptor substrate . Here, 1 ml of crude enzyme was mixed with 2.5 μl of 10 mmol/L FMN‐Na, 300 μl of 0.14 mmol/L NADH, respectively.…”

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

“…For example, the purification of lactate dehydrogenase by Anderson 10 uses affinity by dye-binding, and the purification of Bovine milk α-lactalbumin 7,11 by Boyer uses a nickel-nitriloacetic acid matrix, but no recombinant poly-histidine tag, relying instead on intrinsic affinity of the protein for the resin. Some modern undergraduate laboratory textbooks and publications do implement immobilized metal affinity chromatography on poly-histidine tagged protein targets such as green or red-fluorescent proteins 12,13,14,15 , antibodies 16 , and selected enzymes 17,18,19,20 , even some of unknown function 21 . Arguably, the purification of an enzyme is preferable in the teaching laboratory, because the target can be assayed for activity in subsequent sessions, enriching the experience of "real science" on the part of the student; indeed, these types of laboratory experiences have been published and beneficial outcomes on student learning reported 17,18,20,21 .…”

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

“…For example, the purification of lactate dehydrogenase by Anderson 10 uses affinity by dye-binding, and the purification of Bovine milk α-lactalbumin 7 11 by Boyer uses a nickel-nitriloacetic acid matrix, but no recombinant poly-histidine tag, relying instead on intrinsic affinity of the protein for the resin. Some modern undergraduate laboratory textbooks and publications do implement immobilized metal affinity chromatography on poly-histidine tagged protein targets such as green or red-fluorescent proteins 12 13 14 15 , antibodies 16 , and selected enzymes 17 18 19 20 , even some of unknown function 21 . Arguably, the purification of an enzyme is preferable in the teaching laboratory, because the target can be assayed for activity in subsequent sessions, enriching the experience of "real science" on the part of the student; indeed, these types of laboratory experiences have been published and beneficial outcomes on student learning reported 17 18 20 21 .…”

“…Some modern undergraduate laboratory textbooks and publications do implement immobilized metal affinity chromatography on poly-histidine tagged protein targets such as green or red-fluorescent proteins 12 13 14 15 , antibodies 16 , and selected enzymes 17 18 19 20 , even some of unknown function 21 . Arguably, the purification of an enzyme is preferable in the teaching laboratory, because the target can be assayed for activity in subsequent sessions, enriching the experience of "real science" on the part of the student; indeed, these types of laboratory experiences have been published and beneficial outcomes on student learning reported 17 18 20 21 . And yet, applications of IMAC to enzyme purification in the biochemistry teaching laboratory remain sparse, and the published methods may even presume access to chromatography instrumentation that is typically unavailable for use in the classroom laboratory 20 .…”