Related Experiment Video
Updated: Jun 19, 2026
Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Published on: March 19, 2014
High-level soluble expression, purification and characterization of active human midkine from Escherichia coli
W Kelly Yan1, Marjo Goette, Gabriele Hofmann
1Oncology Protein Sciences, Novartis Institutes for BioMedical Research, 4560 Horton Street, Emeryville, CA 94608, USA. kelly.yan@novartis.com
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.
Insights
Researchers successfully produced high-purity recombinant human Midkine (rhMDK), a crucial heparin-binding growth factor for cancer research. This active rhMDK is vital for advancing cancer drug discovery studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Midkine (MDK) is a heparin-binding growth factor.
- MDK is highly expressed in various cancers.
- MDK is a cysteine-rich protein with five disulfide bonds.
Purpose of the Study:
- To express and purify recombinant human Midkine (rhMDK).
- To confirm the correct disulfide bond formation and biological activity of rhMDK.
- To provide a critical reagent for cancer drug discovery.
Main Methods:
- Recombinant human MDK (rhMDK) expression in Escherichia coli Origami 2 (DE3) strain.
- Two-step purification using heparin affinity and gel filtration chromatography.
- Mass spectrometry for disulfide bond analysis and neurite outgrowth assay for activity confirmation.
Main Results:
- High-level expression of soluble rhMDK achieved.
- Seven milligrams of high-purity rhMDK obtained from a 3 L culture.
- Mass spectrometry confirmed correct disulfide bond linkages.
- Purified rhMDK demonstrated biological activity in a neurite outgrowth assay.
Conclusions:
- The study successfully produced active, purified rhMDK.
- The recombinant protein is suitable for use in cancer drug discovery.
- Active rhMDK is an essential tool for further cancer research.
More Related Videos
07:08Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization
Published on: December 23, 2015
11:20Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014