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