Tsuji Isamu, Mastui Hideki, Ito Tatsuo, Kurokawa Tomofumi, Shintani Yasushi
Biology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan.
Protein Expr Purif. 2011 Dec;80(2):239-45. doi: 10.1016/j.pep.2011.06.015. Epub 2011 Jul 1.
LIGHT is a membrane-bound protein that belongs to the tumor necrosis factor (TNF) superfamily ligands. In this study, we established an effective strategy for producing a bioactive soluble form of LIGHT (sLIGHT), an extracellular region (Ile⁸⁴-Val²⁴⁰) of human LIGHT. Because sLIGHT was expressed as inclusion bodies in Escherichia coli, we investigated reagents that enhance the renaturation of sLIGHT from the inclusion bodies. Interestingly, L-cysteine in the denaturation buffer containing 3.5 M guanidine hydrochloride significantly improved the renaturation efficiency of sLIGHT. The effect of L-cysteine was synergistically enhanced by L-arginine in the refolding buffer. The optimal concentrations of L-cysteine and L-arginine in the denaturation and refolding buffers were 8 mM and 0.8 M, respectively. With these buffers, approximately 90 mg of sLIGHT was purified from 200 g of frozen E. coli cells. sLIGHT thus obtained significantly induced apoptosis in the WiDr human colon adenocarcinoma cell line at nanomolar concentrations, the same amount of sLIGHT that was produced by Sf9 insect cells. These results suggest that L-cysteine in the denaturation buffer enhances the renaturation of recombinant proteins from inclusion bodies in E. coli.
LIGHT是一种膜结合蛋白，属于肿瘤坏死因子（TNF）超家族配体。在本研究中，我们建立了一种有效的策略来生产具有生物活性的可溶性LIGHT（sLIGHT），即人LIGHT的细胞外区域（Ile⁸⁴-Val²⁴⁰）。由于sLIGHT在大肠杆菌中以包涵体形式表达，我们研究了能增强sLIGHT从包涵体中复性的试剂。有趣的是，在含有3.5 M盐酸胍的变性缓冲液中加入L-半胱氨酸可显著提高sLIGHT的复性效率。在复性缓冲液中，L-精氨酸可协同增强L-半胱氨酸的作用。变性缓冲液和复性缓冲液中L-半胱氨酸和L-精氨酸的最佳浓度分别为8 mM和0.8 M。用这些缓冲液，从200 g冷冻大肠杆菌细胞中纯化得到了约90 mg的sLIGHT。如此获得的sLIGHT在纳摩尔浓度下能显著诱导WiDr人结肠腺癌细胞系凋亡，与Sf9昆虫细胞产生的sLIGHT量相同。这些结果表明，变性缓冲液中的L-半胱氨酸可增强大肠杆菌中重组蛋白从包涵体中的复性。
