High-level Soluble Expression, Purification and Characterization of Active Human Midkine from Escherichia coli
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Yan, Wei, Goette, Marjo, Hofmann, Gabriele, Zaror, Isabel and Sim, Bang Janet (2010) High-level Soluble Expression, Purification and Characterization of Active Human Midkine from Escherichia coli. Protein Expression and Purification, 70 (2). pp. 270-276. ISSN 1046-5928
Abstract
Midkine (MDK) belongs to a class of heparin-binding growth factors and is highly expressed in a number of cancers. MDK is a cysteine-rich 13 kDa protein containing five disulfide bonds. In this study, we expressed recombinant human MDK (rhMDK) in Escherichia coli Origami 2 (DE3) strain, which carries a (trxB-/gor522-) double mutation. Soluble rhMDK was expressed at a high level in this strain and the protein was purified by a two-step purification using heparin affinity and gel filtration chromatography. Seven milligrams of rhMDK with high purity were obtained from a 3 L culture. All 10 cysteines were confirmed to be engaged in correct disulfide bond linkages by mass spectrometry analysis. Activity of purified rhMDK was confirmed by a neurite outgrowth assay using rat cerebellar granule cells. Active rhMDK is a critical reagent for cancer drug discovery studies.
| Item Type: | Article |
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| Additional Information: | author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used |
| Keywords: | Human midkine; Disulfide bond; Escherichia coli expression; Affinity purification; Analysis |
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| Date Deposited: | 13 Oct 2015 13:16 |
| Last Modified: | 13 Oct 2015 13:16 |
| URI: | https://oak.novartis.com/id/eprint/1926 |