Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase

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Abstract

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases. Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO). Significant formation of inclusion bodies was noted at the growth temperature of 37 °C, with reduced formation at 30 °C. The addition of the natural biosynthetic precursor of protoporphrin IX, δ-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 °C and purification by nickel–agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 μmol of kynurenine/h/mg of protein (both identical to native human IDO). The protein was homogeneous in terms of electrophoretic analysis. Yields of soluble protein (3–5 mg/L of bacterial culture) and heme content are greater than previously reported.

Section snippets

Materials

δ-Aminolevulinic acid (ALA), ampicillin, ascorbic acid, catalase (bovine), imidazole, isopropyl-β-d-thiogalactopyranoside (IPTG), l-kynurenine, kanamycin, lysozyme, p-dimethylaminobenzaldehyde (p-DMAB), phenylmethylsulfonyl fluoride (PMSF), and l-tryptophan were obtained from Sigma–Aldrich. Bovine serum albumin (BSA) (Fraction V) was obtained from Amersham. Coomassie Blue R250 was purchased from Bio-Rad. DNase and EDTA-free cocktail inhibitor tablets were products of Roche. All other chemicals

Results and discussion

Qiagen, the manufacturer of the expression vector used in these studies, recommends 1 mM IPTG for induction [36]. Littlejohn et al. [33], however, performed a number of trials to determine an optimum IPTG level of 10 μM for the expression of soluble active IDO at 37 °C.

Expression of genetically engineered proteins in bacteria, especially E. coli, often results in the accumulation of insoluble protein aggregates, known as inclusion bodies [37]. Growths at 37 °C utilising 10 μM IPTG may not be

Conclusion

Here, we report an improved expression and purification protocol for recombinant human IDO. Inclusion body formation by EC538 of 6His-IDO was significantly lowered through reduction in growth temperature from 37 to 30 °C. The use of ALA, the biosynthetic precursor of protoporphrin IX, coupled with metal-affinity chromatography and size exclusion chromatography, produced 6His-IDO with a protein to heme ratio of 1:2.2, the same as the native enzyme—better than heme levels previously reported for

Acknowledgments

This work was supported in part by the Australian Postgraduate Award Scheme for a Ph.D. scholarship (Christopher J.D. Austin) and the Australian National Health and Medical Research Council. We are grateful to Dr. Tamantha K. Littlejohn for technical support and assistance. This research has been facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities program.

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