Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification

Abstract

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.

Keywords: 3-Hydroxykynurenine; Kynurenine 3-monooxygenase; Purification; Solubility.

Fig. 1

Kynurenine pathway. Biochemical pathway of tryptophan breakdown, KMO highlighted by blue box (adapted from Crozier-Reabe, 2008 [10]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Protein construct drawings. Cartoon illustrating the composition of each protein construct, (A) truncated hKMO-6 His; (B) full-length hKMO-6×His; (C) full length hKMO-12His-3×FLAG.

Fig. 3

Insoluble flKMO. (A) SDS-PAGE protein gel stained with simplyblue. Lane 1. Soluble fraction following over-expression of full length KMO in bacterial cells. Lane 2. Insoluble fraction, KMO protein (55 kDa) highlighted by arrow. (B) Western blot of the same samples, chemiluminescence detection using Anti-His(c-term)-HRP antibody. Lanes as before. The majority of the KMO protein is present in the insoluble fraction. (C) Cartoon illustrating the flKMO-6His construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Purified trKMO. (A) Chromatogram produced during NiNTA affinity purification of truncate KMO. (B) SDS-PAGE protein gel stained with simplyblue showing HisTrap purification of truncated human KMO protein, (a) Cell-free extract prior to affinity chromatography, (b) flowthrough proteins which did not bind to the column, (c) first column wash, (d) second column wash, (e) third column wash, (f) pure truncated human KMO protein (44 kDa) from the fractions under the chromatogram. (C) Cartoon illustrating the trKMO construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Soluble partially purified KMO-FLAG. (A) Western blot using chemiluminescence detection, anti-FLAG M2 antibody and goat anti-mouse HRP secondary antibody, (a) soluble fraction from over-expressed KMO-12His-FLAG (57 kDa) in bacterial cells. (B) SDS-PAGE gel stained with simplyblue showing anti-FLAG conjugated protein A magnetic bead purification of KMO-12His-FLAG, (a) Cell-free extract prior to purification, (b) flowthrough proteins which did not bind to the beads, (c) first magnetic bead wash, (d) second bead wash, (e) purified KMO-12His (57 kDa) (bold arrow) eluted from the beads by TEV protease has precipitated with two contaminant E. coli proteins (indicated using arrows). (C) Cartoon illustrating the flKMO-12His-FLAG construct. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Kinetic characterisation of KMO. Steady state kinetics for flKMO-6His and purified KMO-12His (FLAG removed during purification) at 37 °C, pH 7. Starting concentrations of kynurenine and NADPH are plotted versus 3-HK produced for each protein and data fitted to the Michaelis–Menten equation (v = V_max.[l-Kyn]/(K_m+[l-Kyn]) using GraphPad Prism4 software.