title: Prokaryotic Expression, Refolding and Purification of High-Purity Mouse Midkine in Escherichia coli.
abstract: 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.
authors: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of China.
authors: Jin
authors: J
authors: Gao
authors: Haixia
authors: H
authors: Wang
mesh_terms: D000818:Animals; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D000078792:Midkine; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; C582105:Mdk protein, mouse; D011994:Recombinant Proteins; D000078792:Midkine