Mattes R
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Semin Thromb Hemost. 2001 Aug;27(4):325-36. doi: 10.1055/s-2001-16886.
Tissue-type plasminogen activator (t-PA) is a valuable thrombolytic agent because of its high affinity to fibrin. When produced in mammalian cell lines, it is glycosylated, a modification that is believed to promote its rapid clearance from the circulation. Bacteria such as Escherichia coli have been tested as alternative expression systems but were not able to express the cDNA of t-PA effectively. The coding sequence for t-PA revealed a significant proportion of AGA and AGG codons, which are rarely used in the coding sequences of E. coli. The argU and argW gene products of E. coli proved to be minor tRNA(arg) species, respectively decoding the very rare triplets AGA/AGG and AGG for arginine. Analysis of genomic fragments from E. coli for both tRNA(arg) genes revealed the presence of defective, cryptic prophages integrated within the impaired tRNA genes. Cloning and supplementation of the limiting tRNA genes argU and argW on helper plasmids improved the translation of the rare AGA and AGG codons. This augmentation improved bacterial growth and enhanced t-PA production in the form of inactive inclusion bodies. This dependence on augmentation of tRNA(arg4) or tRNA(arg5) for improved cell growth and expression was also observed for other genes with a high content of these rare arginine codons. Construction and production of nonglycosylated t-PA in inclusion bodies in E. coli along with improvement of the subsequent renaturation and purification procedures resulted in material comparable to that derived from CHO cells. Deletion of domain-encoding segments yielded various "muteins" of t-PA (e.g., reteplase [rPA]) that could be produced in and activated from the purified inclusion bodies analogously. Furthermore, it was shown that rPA has an extended half-life in the circulation because of its lack of glycosylation and impaired receptor binding capability. rPA was successfully used in various clinical studies. It is a new-generation thrombolytic agent with a longer half-life and can thus be administered more conveniently as a double bolus.
组织型纤溶酶原激活剂（t-PA）因其对纤维蛋白具有高亲和力而成为一种有价值的溶栓剂。在哺乳动物细胞系中产生时，它会被糖基化，这种修饰被认为有助于其从循环中快速清除。诸如大肠杆菌等细菌已被测试作为替代表达系统，但无法有效表达t-PA的cDNA。t-PA的编码序列显示出相当比例的AGA和AGG密码子，而这些密码子在大肠杆菌的编码序列中很少使用。大肠杆菌的argU和argW基因产物被证明是次要的tRNA（arg）种类，分别解码非常罕见的精氨酸三联体AGA/AGG和AGG。对大肠杆菌的tRNA（arg）基因的基因组片段进行分析发现，在受损的tRNA基因中存在整合的缺陷性、隐匿性原噬菌体。在辅助质粒上克隆并补充限制性tRNA基因argU和argW改善了罕见AGA和AGG密码子的翻译。这种增强改善了细菌生长，并以无活性包涵体的形式提高了t-PA的产量。对于其他含有高比例这些罕见精氨酸密码子的基因，也观察到了对tRNA（arg4）或tRNA（arg5）增强以改善细胞生长和表达的依赖性。在大肠杆菌中构建并在包涵体中产生非糖基化t-PA，同时改进后续的复性和纯化程序，得到了与源自CHO细胞的材料相当的物质。缺失编码结构域的片段产生了各种t-PA的“突变体”（例如，瑞替普酶[rPA]），它们可以类似地在纯化的包涵体中产生并激活。此外，研究表明，rPA由于缺乏糖基化和受体结合能力受损，在循环中的半衰期延长。rPA已成功用于各种临床研究。它是一种半衰期更长的新一代溶栓剂，因此可以更方便地作为双剂量推注给药。
