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. (C) 2009 Elsevier Inc. All rights reserved.