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J Biochem Biophys Methods. 2001 Mar 28;48(1):77-84.

A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody.

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Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, 376-8515, Gunma, Japan.


A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK). The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA. An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl. SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.

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