Hofzumahaus Sebastian, Schallmey Anett
Institute of Biotechnology, RWTH Aachen University, Aachen, Germany.
Protein Expr Purif. 2013 Aug;90(2):117-23. doi: 10.1016/j.pep.2013.05.010. Epub 2013 Jun 4.
Elicitins are sterol carrier proteins from the Oomycete genera Phytophthora and Phytium and elicit a hypersensitive response in many economically important plants, in some cases causing a systemic acquired resistance. Their recombinant expression in bacteria is complicated by the presence of three disulfide bonds in the elicitin structure. In consequence, elicitins have so far only been produced in soluble form by isolation from native Phytophthora or Phytium strains or by recombinant expression in the yeast Pichia pastoris. Here, for the first time, we report the soluble expression of the elicitin β-cinnamomin from Phytophthora cinnamomi in Escherichia coli by secretion of the protein into the periplasm. β-Cinnamomin yields have been significantly improved after careful selection of the optimum secretion signal sequence. In total, 17.6 mg β-cinnamomin per liter cell culture have been obtained in shake flasks with the secretion signal sequence of the maltose-binding protein MalE from E. coli. Furthermore, by making use of a C-terminal His-tag, β-cinnamomin purification has been significantly simplified with only one step of immobilized metal ion affinity chromatography yielding protein of high purity (>90%). The established protocol has further been successfully applied to the soluble expression of another elicitin.
激发素是来自疫霉属和腐霉菌属卵菌纲的固醇载体蛋白，能在许多具有重要经济价值的植物中引发超敏反应，在某些情况下还能引起系统获得性抗性。由于激发素结构中存在三个二硫键，其在细菌中的重组表达较为复杂。因此，到目前为止，激发素只能通过从天然疫霉菌或腐霉菌菌株中分离，或通过在巴斯德毕赤酵母中进行重组表达来以可溶形式产生。在此，我们首次报道了通过将来自樟疫霉的激发素β-肉桂疫霉蛋白分泌到周质中，在大肠杆菌中实现其可溶表达。在仔细选择最佳分泌信号序列后，β-肉桂疫霉蛋白的产量得到了显著提高。使用大肠杆菌麦芽糖结合蛋白MalE的分泌信号序列，在摇瓶中每升细胞培养物总共获得了17.6毫克β-肉桂疫霉蛋白。此外，通过利用C端组氨酸标签，仅通过一步固定化金属离子亲和层析就显著简化了β-肉桂疫霉蛋白的纯化过程，得到了高纯度（>90%）的蛋白。所建立的方案已进一步成功应用于另一种激发素的可溶表达。
