Screening of fusion partners for high yield expression and purification of bioactive viscotoxins
Screening of fusion partners for high yield expression and purification of bioactive viscotoxins
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DOI：
10.1016/j.pep.2008.10.003
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发表时间：
2009-03-01
影响因子：
1.6
通讯作者：
Stier, Gunter
中科院分区：
文献类型：
--
作者：
Bogomolovas, Julius;Simon, Bernd;Stier, Gunter
Viscotoxins是在欧洲槲寄生Viscum album中发现的小阳离子蛋白质。它们对植物病原真菌和癌细胞具有高毒性。粘毒素的异源表达将拓宽用于更好地理解其结构和功能的方法的范围，并满足可能的生物制药需求。在这里，我们评估了13种不同的蛋白质作为在大肠杆菌细胞中表达的融合伴侣：His 6标签和His 6标签版本的GB 1，ZZ标签，Z标签，麦芽糖结合蛋白，NusA，谷胱甘肽S-转移酶，硫氧还蛋白，绿色荧光蛋白，以及周质和胞质版本的DsbC和DsbA。与硫氧还蛋白的融合得到最高产量的可溶性粘毒素。用Ni 2+亲和层析捕获His 6-标记的融合蛋白，随后用烟草蚀纹病毒蛋白酶切割。通过酸化裂解混合物进行选择性沉淀，然后进行阳离子交换色谱。该方案从11 μ l培养基中得到5.2mg内脏毒素A3，对应于68%的回收率。质谱分析表明样品纯度高，重组粘毒素中存在三个二硫键。蛋白质的正确折叠通过在均匀15 N标记的样品上记录的异相NMR光谱来确认。使用该方案制备的重组粘毒素对HeLa细胞是有毒的，并且保留了天然蛋白质中发现的亚型B和A3之间的活性差异。(C)2008年爱思唯尔公司All rights reserved.
Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni2+ affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 11 of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins. (C) 2008 Elsevier Inc. All rights reserved.