Macquarie University
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Recombinant human indoleamine 2,3-dioxygenase

posted on 2022-03-28, 19:33 authored by Priambudi Kosim-Satyaputra
Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase that catalyses the first and rate-limiting step in the kynurenine (Kyn) pathway of L-tryptophan (L-Trp)catabolism. Attention has been focused on IDO because the Kyn metabolic pathway is involved in a variety of physiological functions and diseases. In this study the expression and purification of recombinant human indoleamine 2,3-dioxygenase (rhIDO) in E. coli(pQE-9-IDO, pREP4) was investigated in order to obtain high quality enzyme in high yields. This study concluded that optimisation could still be achieved by lowering the growth temperature from 37 °C to 30 °C and reduction of IPTG induction from 100 μMto 10 μM. Better yields of rhIDO were obtained when the expression was carried out under dark conditions without the addition of PMSF, addition of hemin prior to lysis of the cells, increasing the NaCl concentration from 150 mM to 500 mM and lowering the imidazole variation from 10, 30, 40, 65, 80, 90, 190 to 10, 30, 60, 300 mM in the purification through Ni-NTA. Evaluation of the characteristics and stability of rhIDO in order to understand its behaviour as a support towards further studies showed that rhIDO activity and yield under certain conditions of purification were significantly decreased. Further studies concluded that the loss of rhIDO activity was most probably due to the loss of the heme prosthetic group of the enzyme or the non effectiveness of the heme-protein complex. A study with hemin to examine the possibility of re-incorporation of heme showed moderate re-incorporation. It was concluded, however, that keeping the heme intact through the supplementation of hemin prior to lysis is a much better alternative for preserving the activity of the enzyme. Comparison studies between rhIDO and rmIDO showed that the relative catalytic efficiency of rhIDO towards L-Trp (3.73) was higher compared to rmIDO (1.40) whereas the relative catalytic efficiency of rhIDO towards D-Trp (0.02) was lower compared to rmIDO (0.03). CD and thermal melt studies established that rhIDO is less helical (61% helix) than rmIDO (71% helix). The studies also showed that rhIDO has a lower melt temperature (50 ºC) than rmIDO (65 ºC)indicating that rmIDO is more thermostable than rhIDO. The contribution of individual cysteines towards the overall catalytic properties and stability of the rhIDO was evaluated through mutagenesis studies. This was done by comparing the resulting outcomes from the C126A, C286A, C322A and C349A rhIDO mutations towards that of the non-mutated rhIDO. All the mutants exhibited a decrease in specificity towards L-Trp with C322A showing the highest decrease of 78% loss. However, with D-Trp, mutants C126A and C322A showed increased specificity whereC126A showed the highest increase (280%). Mutants C286A and C349A showed decreased activity with C349A having the most (67% loss). This study confirms that the Cys to Ala site-directed mutagenesis contributes to the changes in the kinetics of the mutated rhIDO, and that the cysteine moieties of rhIDO are involved in the normal catalytic function of the enzyme. It is highly likely that the change from Cys to Ala in the mutants changed the conformation of the enzyme, which was a determining factor to the accessibility of the specific substrates to the active site. This change in conformation resulted in diverse kinetics observed.


Table of Contents

1. Introduction -- 2. Expression and purification of indoleamine 2,3-dioxygenase -- 3. Characterisation of indoleamine 2,3-dioxygenase -- 4. Re-incorporation of heme into indoleamine 2,3-dioxygenase -- 5. Mutagenesis of indoleamine 2,3-dioxygenase -- 6. Conclusions and recommendations -- 7. Experimental -- Appendices


Typescript. A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Macquarie University, 2009. Bibliography: leaves 169-205 "December 2009"

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


Thesis (PhD), Macquarie University, Faculty of Science, Dept. of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award


Principal Supervisor

Joanne Jamie

Additional Supervisor 1

Robert Willows


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1 online resource (xxiv, 222 leaves, bound) ill. (some col.), charts

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