Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein
Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein
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DOI：
10.1016/s0378-1119(00)00367-x
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发表时间：
2000-11-27
期刊：
影响因子：
3.5
通讯作者：
Stephens, RS
中科院分区：
文献类型：
--
作者：
Jones, HM;Kubo, A;Stephens, RS
设计、构建了沙眼衣原体L2主要外膜蛋白(MOMP)基因，并在大肠杆菌中进行了表达。对天然氨基酸序列进行反向翻译，得到的核苷酸组合被操纵，以便在保留天然氨基酸序列的同时，沿基因长度均匀分布25个独特的限制性内切酶切点。将合成的基因克隆到T7启动子控制的真核表达载体pET-3a中，对表达产物进行抗原性、细胞定位和功能鉴定。免疫印迹法检测表达产物与天然MOMP的特异性单抗反应。外膜分离证实加工后的蛋白质位于外膜。在大肠杆菌中表达并存在于外膜中的MOMP被证明具有广泛的扩散孔功能。该系统提供了以纯化形式或膜结合蛋白的形式生产易于修饰的MOMP的手段，从而促进了对其功能、结构和抗原性的研究。(C)2000 Elsevier Science B.V.保留所有权利。
A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli. The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence. The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function. Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot. Outer membrane fractionation confirmed that the processed protein was located in the outer membrane. MOMP expressed in E. coli and present in the outer membrane was shown to function as a general diffusion porin. This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties. (C) 2000 Elsevier Science B.V. All rights reserved.