High-level Soluble Expression, Purification and Characterization of Active Human Midkine from Escherichia coli. Protein expression and purification [Protein Expr Purif] Journal article | | Title | High-level Soluble Expression, Purification and Characterization of Active Human Midkine from Escherichia coli. | | Author(s) | Yan K, Göette M, Hofmann G, Zaror I, Sim J | | Institution | Oncology Protein Sciences, Novartis Institutes for BioMedical Research, 4560 Horton St. Emeryville, CA 94608, USA. | | Source | Protein Expr Purif 2009 Oct 30. | | 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(-)/gor(522-)) 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. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19883768 |
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