E. coli expression and purification of human and cynomolgus IL-15 |
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Authors: | Alison Ward Malcolm Anderson Robert I Craggs Justine Maltby Caroline Grahames Rick A Davies Donna Finch Debbie Pattison Heather Oakes Philip R Mallinder |
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Institution: | aAstraZeneca R&D Charnwood, Bioscience Department, Bakewell Road, Loughborough LE11 5RH, UK;bAstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK;cMedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, UK |
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Abstract: | The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)6-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His)6-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3− CD8+ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography. |
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Keywords: | Interleukin-15 IL-15 CD69 upregulation CTLL2 Folding Folding screen |
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