Prokaryotic Expression, Refolding and Purification of High-Purity Mouse Midkine in Escherichia coli.

To evaluate the clinic safety of human Midkine as an articular protective agent, recombinant mouse Midkine (rmMK) was prepared in prokaryotic system for the pre-clinic long-term studies in mice. The open reading frame of mouse Midkine (mMK) was sub-cloned onto expression vector pET30a (+) and transformed into Escherichia coli BL21 (DE3) strain line. The rmMK protein, with a Met fused at N terminus of native mMK for expression initiating, proved to be expressed in inclusion bodies and turned out to be soluble post-denaturation and renaturation. The soluble rmMK was purified successfully with ion exchange and affinity chromatography and characterised good enough to meet the requirements for animal use. Eventually, 13.2-mg rmMK with high quality and bioactivity was obtained from 1 L LB culture, and the total recovery was 11.4%. The present work laid a good foundation for pilot- or large-scale production of rmMK in prokaryotic system.