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

Protein Expression and Purification
Christopher J D AustinJoanne F Jamie

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 degrees C, with reduced formation at 30 degrees C. The addition of the natural biosynthetic precursor of protoporphrin IX, delta-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 degrees 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 micromol 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.

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Citations

Feb 9, 2010·Amino Acids·Christopher Jonathan Daraius AustinH J Ball
Mar 26, 2013·Journal of Medicinal Chemistry·Raffaele PasceriChristopher J Moody
Dec 3, 2005·Analytical Biochemistry·Azadeh MatinJoanne F Jamie
Jun 12, 2013·Biochemical and Biophysical Research Communications·Christopher J D AustinJoanne F Jamie
Jun 9, 2016·European Journal of Medicinal Chemistry·Saurav PaulDebasis Manna
Dec 3, 2016·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Emma L Raven
Apr 21, 2006·Archives of Biochemistry and Biophysics·Anne PoljakGeorge A Smythe
Sep 15, 2018·Journal of Drug Targeting·Yuandi ZhaoHongmin Liu
Apr 23, 2014·Dalton Transactions : an International Journal of Inorganic Chemistry·Christopher J D AustinLouis M Rendina
Mar 27, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Chuan ZhouZhiqiang Feng
Feb 7, 2020·Proceedings of the National Academy of Sciences of the United States of America·Giada MondanelliUrsula Grohmann
Jul 26, 2015·Acta Crystallographica Section B, Structural Science, Crystal Engineering and Materials·Andrea CarlettaSalvatore Guccione
Mar 13, 2014·Organic & Biomolecular Chemistry·Catarina CarvalhoChristopher J Moody
Jun 15, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Hui WenLi Sheng
Apr 27, 2021·Bioorganic & Medicinal Chemistry·Rossella Capochiani de IudicibusLai-Ming Ching

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