Sequence analysis and recombinant expression of 28-kilodalton Treponema pallidum subsp pallidum rare outer membrane protein (Tromp2)
Sequence analysis and recombinant expression of 28-kilodalton Treponema pallidum subsp pallidum rare outer membrane protein (Tromp2)
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DOI：
10.1128/jb.179.4.1230-1238.1997
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发表时间：
1997-02-01
影响因子：
3.2
通讯作者：
Lovett, MA
中科院分区：
文献类型：
--
作者：
Champion, CI;Blanco, DR;Lovett, MA
在这项研究中，我们报告的克隆，测序和表达的基因编码的28 kDa梅毒螺旋体亚种，梅毒罕见的外膜蛋白（TROMP），指定Tromp 2。tromp 2基因编码242个氨基酸的前体蛋白，包括以Leu-Ala-Ala的I型信号肽酶切割位点结束的24个氨基酸的推定信号肽。218个氨基酸的成熟蛋白质的计算分子量为24，759，计算pI为7.3。预测的Tromp 2的二级结构显示了外膜蛋白典型的两亲性β-折叠的九个跨膜区段。利用严格调控的T7 RNA聚合酶表达载体，表达了含有天然信号肽的重组Tromp 2（rTromp 2）。在高水平表达条件下，rTromp 2仅与大肠杆菌外膜分离。pallidum中，28 kDa的Tromp 2蛋白主要存在于去污剂相中。pallidum，去除外周相关膜蛋白的条件，证明Tromp 2是一种完整的膜蛋白。E.表达rTromp 2的大肠杆菌细胞显示特异性表面抗体结合。这些发现表明，Tromp 2是一种跨膜外膜蛋白，这是在T.苍白球
In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp, pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector, Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane, Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations, Following Triton X-114 extraction and phase separation of T. pallidum, the 28 kDa Tromp2 protein was detected prominently in the detergent phase, Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.