Full Text:   <1708>

CLC number: Q81

On-line Access: 

Received: 2008-12-08

Revision Accepted: 2009-01-16

Crosschecked: 2009-01-16

Cited: 10

Clicked: 3493

Citations:  Bibtex RefMan EndNote GB/T7714

-   Go to

Article info.
1. Reference List
Open peer comments

Journal of Zhejiang University SCIENCE B 2009 Vol.10 No.2 P.79~86


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

Author(s):  Zhong-hui ZHANG, Li-juan DU, Di XIANG, Shun-ying ZHU, Ming-yuan WU, Hui-li LU, Yan YU, Wei HAN

Affiliation(s):  Laboratory of Regenoromics, School of Pharmacy; more

Corresponding email(s):   weihan@sjtu.edu.cn

Key Words:  Expression, Purification, Human midkine, Escherichia coli

Share this article to: More |Next Article >>>

Zhong-hui ZHANG, Li-juan DU, Di XIANG, Shun-ying ZHU, Ming-yuan WU, Hui-li LU, Yan YU, Wei HAN. Expression and purification of bioactive high-purity human midkine in Escherichia coli[J]. Journal of Zhejiang University Science B, 2009, 10(2): 79~86.

@article{title="Expression and purification of bioactive high-purity human midkine in Escherichia coli",
author="Zhong-hui ZHANG, Li-juan DU, Di XIANG, Shun-ying ZHU, Ming-yuan WU, Hui-li LU, Yan YU, Wei HAN",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Expression and purification of bioactive high-purity human midkine in Escherichia coli
%A Zhong-hui ZHANG
%A Li-juan DU
%A Shun-ying ZHU
%A Ming-yuan WU
%A Hui-li LU
%A Yan YU
%A Wei HAN
%J Journal of Zhejiang University SCIENCE B
%V 10
%N 2
%P 79~86
%@ 1673-1581
%D 2009
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B0820385

T1 - Expression and purification of bioactive high-purity human midkine in Escherichia coli
A1 - Zhong-hui ZHANG
A1 - Li-juan DU
A1 - Shun-ying ZHU
A1 - Ming-yuan WU
A1 - Hui-li LU
A1 - Yan YU
A1 - Wei HAN
J0 - Journal of Zhejiang University Science B
VL - 10
IS - 2
SP - 79
EP - 86
%@ 1673-1581
Y1 - 2009
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B0820385

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.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1] Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72(1-2):248-254.

[2] Choudhuri, R., Zhang, H.T., Donnini, S., Ziche, M., Bicknell, R., 1997. An angiogenic role for the neurokines midkine and pleiotrophin in tumorigenesis. Cancer Res., 57(9): 1814-1819.

[3] Dansithong, W., Paul, S., Kojima, Y., Kamiya, K., Shinozawa, T., 2001. A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody. J. Biochem. Biophys. Methods, 48(1):77-84.

[4] Fabri, L., Maruta, H., Muramatsu, H., Muramatsu, T., Simpson, R.J., Burgess, A.W., Nice, E.C., 1993. Structural characterization of native and recombinant forms of the neurotrophic cytokine MK. J. Chromatogr. A, 646(1): 213-225.

[5] Garver, R.I., Chan, C.S., Milner, P.G., 1993. Reciprocal expression of pleiotrophin and midkine in normal versus malignant lung tissues. Am. J. Respir. Cell Mol. Biol., 9(5):463-466.

[6] Garver, R.I., Radford, D.M., Donis-Keller, H., Wick, M.R., Milner, P.G., 1994. Midkine and pleiotrophin expression in normal and malignant breast tissue. Cancer, 74(5): 1584-1590.

[7] Iwasaki, W., Nagata, K., Hatanaka, H., Inui, T., Kimura, T., Muramatsu, T., Yoshida, K., Tasumi, M., Inagaki, F., 1997. Solution structure of midkine, a new heparin-binding growth factor. EMBO J., 16(23):6936-6946.

[8] Kadomatsu, K., Muramatsu, T., 2004. Midkine and pleiotrophin in neural development and cancer. Cancer Lett., 204(2):127-143.

[9] Kadomatsu, K., Hagihara, M., Akhter, S., Fan, Q.W., Muramatsu, H., Muramatsu, T., 1997. Midkine induces the transformation of NIH3T3 cells. Br. J. Cancer, 75(3): 354-359.

[10] Kaneda, N., Talukder, A.H., Nishiyama, H., Koizumi, S., Muramatsu, T., 1996. Midkine, a heparin-binding growth/differentiation factor, exhibits nerve cell adhesion and guidance activity for neurite outgrowth in vitro. J. Biochem., 119(6):1150-1156.

[11] Kurtz, A., Schulte, A.M., Wellstein, A., 1995. Pleiotrophin and midkine in normal development and tumor biology. Crit. Rev. Oncog., 6(2):151-177.

[12] Maruta, H., Bartlett, P.F., Nurcombe, V., Nur-E-Kamal, M.S., Chomienne, C., Muramatsu, T., Muramatsu, H., Fabri, L., Nice, E., Burgess, A.W., 1993. Midkine (MK), a retinoic acid (RA)-inducible gene product, produced in E. coli acts on neuronal and HL60 leukemia cells. Growth Factors, 8(2):119-134.

[13] Mitsiadis, T.A., Salmivirta, M., Muramatsu, T., Muramatsu, H., Rauvala, H., Lehtonen, E., Jalkanen, M., Thesleff, I., 1995. Expression of the heparin-binding cytokines, midkine (MK) and HB-GAM (pleiotrophin) is associated with epithelial-mesenchymal interactions during fetal development and organogenesis. Development, 121(1): 37-51.

[14] Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65(1-2):55-63.

[15] Muramatsu, H., Shirahama, H., Yonezawa, S., Maruta, H., Muramatsu, T., 1993. Midkine, a retinoic acid-inducible growth/differentiation factor: immunochemical evidence for the function and distribution. Dev. Biol., 159(2): 392-402.

[16] Muramatsu, T., 1994. The midkine family of growth/ differentiation factors. Dev. Growth Differ., 36(1):1-8.

[17] Murasugi, A., Tohma-Aiba, Y., 2003. Production of native recombinant human midkine in the yeast, Pichia pastoris. Protein Expr. Purif., 27(2):244-252.

[18] Murasugi, A., Tohma-Aiba, Y., Asami, Y., 2000. Production of recombinant human midkine in yeast, Pichia pastoris. J. Biosci. Bioeng., 90(4):395-399.

[19] Nakagawara, A., Milbrandt, J., Muramatsu, T., Deuel, T.F., Zhao, H., Cnaan, A., Brodeur, G.M., 1995. Differential expression of pleiotrophin and midkine in advanced neuroblastomas. Cancer Res., 55(8):1792-1797.

[20] Nakamoto, M., Matsubara, S., Miyauchi, T., Obama, H., Ozawa, M., Muramatsu, T., 1992. A new family of heparin binding growth/differentiation factors: differential expression of the midkine (MK) and HB-GAM genes during mouse development. J. Biochem., 112(3): 346-349.

[21] Oakley, B.R., Kirsch, D.R., Morris, N.R., 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem., 105(1):361-363.

[22] Ochs, D.C., McConkey, E.H., Sammons, D.W., 1981. Silver stains for proteins in polyacrylamide gels: a comparison of six methods. Electrophoresis, 2(5-6):304-307.

[23] Ohta, S., Muramatsu, H., Senda, T., Zou, K., Iwata, H., Muramatsu, T., 1999. Midkine is expressed during repair of bone fracture and promotes chondrogenesis. J. Bone Miner. Res., 14(7):1132-1144.

[24] Ratovitski, E.A., Kotzbauer, P.T., Milbrandt, J., Lowenstein, C.J., Burrow, C.R., 1998. Midkine induces tumor cell proliferation and binds to a high affinity signaling receptor associated with JAK tyrosine kinases. J. Biol. Chem., 273(6):3654-3660.

[25] Sammons, D.W., Adams, L.D., Nishizawa, E.E., 1981. Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis, 2(3):135-141.

[26] Seddon, A.P., Hulmes, J.D., Decker, M.M., Kovesdi, I., Fairhurst, J.L., Backer, J., Dougher-Vermazen, M., Bohlen, P., 1994. Refolding and characterization of human recombinant heparin-binding neurite-promoting factor. Protein Expr. Purif., 5(1):14-21.

[27] Yoshida, Y., Goto, M., Tsutsui, J., Ozawa, M., Sato, E., Osame, M., Muramatsu, T., 1995. Midkine is present in the early stage of cerebral infarct. Dev. Brain Res., 85(1):25-30.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - Journal of Zhejiang University-SCIENCE