De Paula V S, Silva F H S, Francischetti I M B, Monteiro R Q, Valente A P
Campus Xerém, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 25245-390, Brazil; Centro de Biologia Estrutural e Bioimagem, Rio de Janeiro, 21941-920, Brazil.
Centro de Biologia Estrutural e Bioimagem, Rio de Janeiro, 21941-920, Brazil; Instituto de Bioquímica Médica, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, 21941-920, Brazil.
Protein Expr Purif. 2017 Nov;139:49-56. doi: 10.1016/j.pep.2017.07.012. Epub 2017 Jul 19.
Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D H and 2D N-H NMR analysis yielded high quality 2D N-H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions.
艾索拉利司是在肩突硬蜱唾液中发现的一种抗凝血蛋白。艾索拉利司含有2个类库尼茨结构域，可作为抑制组织因子（TF）/因子VIIa（FVIIa）的支架与因子Xa或因子X结合。然而，与组织因子途径抑制剂（TFPI）不同，艾索拉利司不与FXa的活性位点裂隙结合。相反，复合物的形成是由FXa肝素结合外位点介导的。由于其强大且持久的抗血栓活性，艾索拉利司是抗凝治疗的一种有前景的药物。尽管对艾索拉利司已有大量功能研究，但尚未获得其原子分辨率的三维结构。我们使用pET32载体成功表达了一种TRX-His-艾索拉利司融合蛋白。通过结合镍-氮三乙酸（Ni-NTA）色谱法、肠激酶蛋白酶切割和反相高效液相色谱（RP-HPLC），我们纯化了用于核磁共振（NMR）研究的同位素标记的艾索拉利司。一维氢（1D H）和二维氮-氢（2D N-H）NMR分析产生了高质量的二维氮-氢异核单量子相干（2D N-H HSQC）谱，表明重组蛋白已折叠。这些研究是通过NMR获得艾索拉利司高分辨率结构信息的第一步，有助于研究艾索拉利司与凝血因子相互作用的分子基础。
