Construction and expression of a recombinant antibody-targeted plasminogen activator.
Construction and expression of a recombinant antibody-targeted plasminogen activator.
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DOI：
10.1073/pnas.84.19.6904
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发表时间：
1987-10
影响因子：
11.1
通讯作者：
Janet M. Schnee;Marschall S. Runge;G. R. Matsueda;Norman W. HUDSONt;J. G. Seidmant;Edgar Haber;And;Thomas Quertermous
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文献类型：
--
作者：
Janet M. Schnee;Marschall S. Runge;G. R. Matsueda;Norman W. HUDSONt;J. G. Seidmant;Edgar Haber;And;Thomas Quertermous
组织型纤溶酶原激活剂（t-PA）与纤维蛋白β链特异性单克隆抗体（抗纤维蛋白59 D8）的共价连接导致比单独的t-PA更特异和更有效的血栓溶解剂。为了提供该杂合分子的现成来源并允许定制活性部分以获得最佳活性，我们设计了59 D8-t-PA缀合物的重组形式。克隆重排的59 D8重链基因，并将其与编码t-PA的γ 2b恒定区和催化β链的一部分的序列组合在表达载体pSV 2gpt中。将该构建体转染到源自59 D8杂交瘤的重链缺失变体细胞中。用亲和层析法纯化重组蛋白，并用电泳转移印迹法进行分析。这些揭示了65-kDa重链-t-PA融合蛋白，其以170-kDa二硫键连接的二聚体的形式与59 D8轻链结合分泌。显色底物测定显示融合蛋白具有天然t-PA的70%的溶肽活性，并且与t-PA一样有效地激活纤溶酶原。在竞争性结合测定中，显示重构抗体具有与天然59 D8相似的结合特征。因此，通过重组技术，我们已经产生了一种能够高亲和力结合纤维蛋白和激活纤溶酶原的杂合蛋白。
Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin beta chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent than t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, we have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, we have produced a hybrid protein capable of high-affinity fibrin binding and plasminogen activation.