title: High-level soluble expression, purification and characterization of active human midkine from Escherichia coli.
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 was 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.
authors.affiliation: Oncology Protein Sciences, Novartis Institutes for BioMedical Research, 4560 Horton Street, Emeryville, CA 94608, USA. kelly.yan@novartis.com
authors.forename: W Kelly
authors.initials: WK
authors.lastname: Yan
authors.forename: Marjo
authors.initials: M
authors.lastname: Goette
authors.forename: Gabriele
authors.initials: G
authors.lastname: Hofmann
authors.forename: Isabel
authors.initials: I
authors.lastname: Zaror
authors.forename: Janet
authors.initials: J
authors.lastname: Sim
mesh_terms: D002531:Cerebellum / Q000166:cytology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D000078792:Midkine; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: C066254:MDK protein, human; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D000078792:Midkine