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首页> 外文期刊>Journal of bacteriology >Renaturation of Recombinant Treponema pallidum Rare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation
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Renaturation of Recombinant Treponema pallidum Rare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation

机译：重组梅毒螺旋体稀有外膜蛋白1的复性成三聚体，疏水性和多孔蛋白活性构象。

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We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1’s hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 ? cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 ? cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1 showed a marked loss in alpha-helical secondary structure of renatured rTromp1 compared to the nonrenatured form. Finally, renatured rTromp1, but not the nonrenatured form, showed porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-active conformation similar to that of the native protein.
机译：我们以前曾观察到，天然的梅毒螺旋体稀有外膜蛋白1（Tromp1）具有疏水性并具有孔蛋白活性，而Tromp1的重组形式则不具备这些特性。在这项研究中，我们表明这些特性是由构象决定的，并且可以通过适当重组重组Tromp1来复制。发现使用Triton X-114从整个生物中提取的天然Tromp1（而非47 kDa脂蛋白）在8 M尿素中处理后失去了疏水性，这表明Tromp1的疏水性取决于构象。天然Tromp1通过快速高效液相色谱（FPLC）从完整生物的0.1％Triton X-100提取物中纯化得到，并显示在平面脂质双层中具有孔蛋白活性。纯化的天然Tromp1与11？的交联研究。交联剂显示出与二聚体和三聚体一致的寡聚形式。为了对重组Tromp1（rTromp1）进行复性研究，在大肠杆菌中表达了31,109-Da的无信号构建体，并通过FPLC纯化。将FPLC纯化的rTromp1在8 M尿素中变性，然后在透析期间在存在0.5％Zwittergent 3,14的情况下进行变性，以去除尿素。使变性的rTromp1通过事先用已知分子量标准校准的Sephacryl S-300凝胶排阻色谱柱。尽管所有未变性的rTromp1均从碳酸酐酶蛋白质标准品（29 kDa）的位置洗脱，而所有变性的rTromp1均在磷酸酶 b 蛋白质标准品（97 kDa）的位置洗脱。三聚体构象。通过使用11？确认三聚化。交联剂，其二聚体和三聚体均与天然Tromp1相似。 Triton X-114相分离显示，所有变性的rTromp1相，但没有一个未变性的rTromp1相，仅与疏水性去污剂相分离，类似于天然Tromp1。与未变性的rTromp1相比，未变性和已变性的rTromp1的圆二色性显示出α-螺旋二级结构的明显损失。最后，经过变性的rTromp1，而非未经变性的形式，在平面液体双层中显示孔蛋白活性。这些结果表明，rTromp1的正确折叠会导致三聚体，疏水性和孔蛋白活性构象，类似于天然蛋白。

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