The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity.
The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity.
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DOI：
10.1186/1472-6750-9-43
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发表时间：
2009-05-14
影响因子：
3.5
通讯作者：
Ranson M
中科院分区：
文献类型：
--
作者：
Cochran BJ;Gunawardhana LP;Vine KL;Lee JA;Lobov S;Ranson M
纤溶酶原激活物抑制剂2型（派-2，SERPINB 2）是尿激酶纤溶酶原激活物（uPA）的不可逆、特异性抑制剂。由于uPA在癌细胞表面的过表达与恶性肿瘤有关，因此已经提出通过外源性重组派-2靶向uPA作为潜在癌症治疗的基础。为此，需要保持这种靶向能力的高纯度蛋白质的可再现产量。在此，我们验证了在体外使用重组6 × His标记的派-2，其缺乏C和D α螺旋之间的螺旋内环（派-2 Δ CD环）用于这些目的。结果表明，从pQE 9载体系统中表达和纯化的派-2 Δ CD-环比以前使用的pET 15 b系统更容易纯化。此外，派-2 Δ CD-loop表达产物的产量和纯度均高于相同条件下表达和纯化的野生型派-2。重要的是，CD环的缺失对溶液相和细胞表面uPA的抑制或对受体结合的uPA从细胞表面的清除没有影响。此外，uPA：派-2 Δ CD环复合物与内吞作用受体极低密度脂蛋白受体（VLDLR）的结合动力学（KD ~5 nM）与先前发表的uPA：派-2复合物相似。我们证明CD环对于细胞uPA抑制和细胞表面清除（内吞作用）的目的是多余的，因此适合于开发抗uPA靶向癌症治疗剂。
Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use in vitro of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes. We show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (KD ~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes. We demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.
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影响因子：
8.8
作者：
通讯作者：
--
影响因子：
11.4
作者：
BELIN, D;WOHLWEND, A;VASSALLI, JD
通讯作者：
VASSALLI, JD
DOI：
10.1107/s0907444906052024
发表时间：
2007-03-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Carson, Mike;Johnson, David H.;DeLucas, Lawrence J.
通讯作者：
DeLucas, Lawrence J.
影响因子：
10.3
作者：
Look, MP;van Putten, WLJ;Foekens, JA
通讯作者：
Foekens, JA
影响因子：
7.5
作者：
Muehlenweg, B;Guthaus, E;Magdolen, V
通讯作者：
Magdolen, V