Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75
Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75
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DOI：
10.1016/j.jbiotec.2008.04.012
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发表时间：
2008-06-30
影响因子：
4.1
通讯作者：
De Greve, Henri
中科院分区：
文献类型：
--
作者：
Tran, Thao;Buesher, Philippe;De Greve, Henri
不变表面糖蛋白ISG 75是存在于血流形式锥虫表面的跨膜糖蛋白。本研究描述了表达和纯化的N-末端胞外结构域的ISG 75，一个新的目标，为锥虫病的诊断测试的发展。为了促进细胞质中二硫键的形成，在硫氧还蛋白还原酶、谷胱甘肽氧化还原酶双突变体大肠杆菌菌株中表达编码来自布氏冈比亚锥虫的ISG 75的1287-bp cDNA片段。一个辅助质粒pRIL，提供的argI，ileY，和leuW的tRNA，是必要的ISG 75 mRNA的有效异源翻译。重组双标签（链霉亲和素和组氨酸）ISG 75通过两步亲和层析纯化。在缓冲溶液中添加L-谷氨酸和L-精氨酸对于在纯化期间稳定蛋白质是至关重要的。纯化的可溶性蛋白，其特征在于通过圆二色谱，反相高压液相色谱和质谱。它具有α-螺旋折叠构象，是均匀和纯的（99%）。此外，布氏锥虫感染的动物的血清特异性地识别该重组ISG 75;并且针对重组ISG 75产生的兔抗血清检测寄生虫制剂中的锥虫亚属的所有种属。(C)2008 Elsevier B. V.保留所有权利。
The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilitate disulfide formation in the cytoplasm, a 1287-bp cDNA fragment encoding ISG75 from Trypanosoma brucei gambiense was expressed in a thioredoxin reductase, glutathione oxidoreductase double mutant Escherichia coli strain. An accessory plasmid pRIL, providing the argI, ileY, and leuW tRNAs, was necessary for efficient heterologous translation of the ISG75 mRNA. The recombinant double-tagged (streptavidine and histidine) ISG75 was purified by two-step affinity chromatography. Addition of L-glutamic acid and L-arginine in the buffer solutions was crucial to stabilise the protein during purification. The purified soluble protein was characterised by circular dichroism spectroscopy, reverse-phase high pressure liquid chromatography and mass spectrometry. It has an alpha-helical folded conformation, is homogeneous and pure (99%). Furthermore, sera of Trypanosoma brucei-infected animals specifically recognise this recombinant ISG75; and rabbit antiserum raised against the recombinant ISG75 detects all species of the Trypanozoon subgenus in parasite preparations. (C) 2008 Elsevier B.V. All rights reserved.