Expression and purification of bioactive high-purity human midkine in Escherichia coli

Abstract

Abstract:Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor. In the present work, rhMK was expressed in Escherichia coli (E. coli) BL21 (DE3). The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG). After sonication, midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC). The purified rhMK enhanced the proliferation of NIH3T3 cells.

Eukaryotic Expression and Purification of Native Form of Mouse Midkine from Pichia pastoris

“…In our regenerative laboratory, an NIH3T3 proliferation-promoting activity had also been developed to evaluate the biological activity of purified rhMK [15]. Since rhMK is aimed to be developed to accelerate cartilage regeneration, biological activity of rhMK on cartilage cells is more practical and meaningful.…”

The Therapeutic Effect of rhMK on Osteoarthritis in Mice, Induced by Destabilization of the Medial Meniscus

“…29) Briefly, isopropylthio-b-D-galactoside (IPTG) was used to induce the expression of rhMK at the final concentration of 1 mM. The midkine was efficiently expressed as the form of inclusion bodies.…”

The Denaturing and Renaturing are Critical Steps in the Purification of Recombinant Protein in Prokaryotic System

“…The recombinant protein in supernatant of the lyzed bacteria can be purified by 2-steps of ionexchange chromatography to yield high-output biological functional proteins. However, some other proteins, such as recombinant human Midkine (rhMK), 8 recombinant mouse monokine induced by IFN-γ (rmMig) 9 , recombinant human Reg 4 (rhReg-4) 10 or recombinant human Chemerin (rhChemerin), 11 easily form inclusion bodies in E. coli, hence, necessary denaturing and renaturing steps must be applied. In some other cases, like recombinant murine CXCL14 (rmCXCL14), 12 full length protein is hard to be purified, and an additional tag will help to get final products with high yield and high purity.…”

“…After induction, rhMidkine mainly exists in inclusion bodies of the cell lysates. 22 Mouse CXCL14/BRAK is a monocyte-selective chemokine which is expressed in almost all normal tissues. The mouse cxcl14 mRNA encodes 99 amino acids, in which N-terminal 22 amino acids serve as a signal peptide which is cleaved as the protein is secreted.…”