Midkine (MK), a retinoic acid (RA)-inducible gene product, produced in E. coli acts on neuronal and HL60 leukemia cells.
We have shown previously that (i) retinoic acid (RA), an anti-neoplastic agent, activates the midkine (MK) gene in mammalian embryonic carcinoma cells, and that (ii) the MK of 118 amino acids, purified from L cells, induces neurite outgrowth of mammalian embryonic brain cells. In this paper, we describe an unconventional strategy for the purification of a fully active MK from E. coli with a high yield. The MK was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein. The MK fusion protein extracted from the bacterial inclusion bodies with guanidine-HCl was renatured, refolded slowly and cleaved by thrombin at the site where the GST links to the MK. The purified free MK, like RA, induced neurite outgrowth from central neurons of the mouse spinal cord, and suppressed the growth of human HL60 leukemia cells in vitro. Unlike RA, however, the MK did not induce granulocytic differentiation of HL60 cells. Furthermore, the MK supported the survival of an NGF-insensitive sensory neuron subpopulation(s) from chicken embryo dorsal root ganglion. Thus, the actions of the MK and leukemia inhibitory factor (LIF) are surprisingly similar. There is no sequence similarity between MK and LIF, however, and unlike MK, LIF production does not appear to be RA-inducible.
Maruta
H
H
Melbourne Tumor Biology Branch, Ludwig Institute for Cancer Research, Victoria, Australia.
Bartlett
P F
PF
Nurcombe
V
V
Nur-E-Kamal
M S
MS
Chomienne
C
C
Muramatsu
T
T
Muramatsu
H
H
Fabri
L
L
Nice
E
E
Burgess
A W
AW
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016207:Cytokines; D004622:Embryo, Mammalian; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D051379:Mice; D000078792:Midkine; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure; D009475:Neurons, Afferent / Q000166:cytology* / Q000187:drug effects; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013116:Spinal Cord / Q000166:cytology; D013724:Teratoma; D013917:Thrombin / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D016207:Cytokines; D009414:Nerve Growth Factors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000078792:Midkine; D014212:Tretinoin; D005982:Glutathione Transferase; D013917:Thrombin