High-level soluble expression, purification and characterization of active human midkine from Escherichia coli.
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.
Yan
W Kelly
WK
Oncology Protein Sciences, Novartis Institutes for BioMedical Research, 4560 Horton Street, Emeryville, CA 94608, USA. kelly.yan@novartis.com
Goette
Marjo
M
Hofmann
Gabriele
G
Zaror
Isabel
I
Sim
Janet
J
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
C066254:MDK protein, human; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D000078792:Midkine