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
Melbourne Tumor Biology Branch, Ludwig Institute for Cancer Research, Victoria, Australia.
Growth Factors. 1993;8(2):119-34. doi: 10.3109/08977199309046932.
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.
（i）维甲酸（RA），一种抗肿瘤药物，可在哺乳动物胚胎癌细胞中激活中期因子（MK）基因；以及（ii）从L细胞中纯化得到的118个氨基酸的MK可诱导哺乳动物胚胎脑细胞的神经突生长。在本文中，我们描述了一种从大肠杆菌中高产纯化出具有完全活性的MK的非常规策略。MK在大肠杆菌中作为谷胱甘肽S-转移酶（GST）融合蛋白过量表达。用盐酸胍从细菌包涵体中提取的MK融合蛋白经复性、缓慢重折叠，并在GST与MK连接的位点被凝血酶切割。纯化后的游离MK与RA一样，可诱导小鼠脊髓中枢神经元的神经突生长，并在体外抑制人HL60白血病细胞的生长。然而，与RA不同的是，MK不会诱导HL60细胞的粒细胞分化。此外，MK可支持鸡胚背根神经节中对神经生长因子（NGF）不敏感的感觉神经元亚群的存活。因此，MK与白血病抑制因子（LIF）的作用惊人地相似。然而，MK与LIF之间没有序列相似性，而且与MK不同，LIF的产生似乎不是RA诱导的。
