Expression and purification of bioactive high-purity human midkine in Escherichia coli.
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-beta-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.
Zhang
Zhong-hui
ZH
Laboratory of Regenoromics, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Du
Li-juan
LJ
Xiang
Di
D
Zhu
Shun-ying
SY
Wu
Ming-yuan
MY
Lu
Hui-li
HL
Yu
Yan
Y
Han
Wei
W
D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D000078792:Midkine; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D016207:Cytokines; D011994:Recombinant Proteins; D000078792:Midkine