Development of high-yield in vitro and in vivo protein expression systems based on Leishmania tarentolae

重组蛋白的产生是生命科学的一个关键方法，特别是生物技术产业。尽管有超过30年的历史，重组形式的真核蛋白的生产仍然存在问题。在很大程度上，这是由于所有可用的真核蛋白表达系统都利用内源性和高度调节的RNA聚合酶II进行异源基因转录的事实。转录控制机制的复杂性阻碍了工程化真核生物可重复地产生大量重组多肽。为了建立一个可靠的、高产量的真核表达系统，我们利用了快速生长的非致病性动质体利什曼原虫。动质体与其他真核生物不同的特征是能够翻译由RNA聚合酶I或甚至外源聚合酶产生的信使RNA，这是由于转录与RNA加工的天然解偶联。在最成功的实施方式中，将异源基因在环状附加体上递送到鞑靼乳杆菌菌株中。尽管该系统显示每升递送高达300mg的重组蛋白，但其遭受强烈的克隆变异。初步结果表明，这可能与利什曼原虫细胞中附加体元件的行为有关。了解环状和线性附加体的结构动力学、遗传和转录信息对于构建新一代表达载体和开发抑制携带致病菌株耐药基因的附加体的策略是必不可少的。在一个互补的方法，我们计划开发一个高产量的体外翻译系统的基础上提取的Ltarentolae。这样的系统预期比其他体外翻译系统更稳健且便宜得多。

Generation of recombinant proteins is a key methodology for Life Science in general and the biotechnological industry in particular. Despite over 30 year long history production of eukaryotic proteins in recombinant form remains problematic. To a significant extent this is due to the fact tfiat all available eukaryotic protein expression Systems utilize the endogenous and highiy regulated RNA polymerase II for heterologous gene transcription. The complexity of the transcription control machinery precludes engineering eukaryotes ttiat reproducibly produce large amounts of recombinant polypeptides. In order to develop a reliable and high-yield eukaryotic expression System we exploited the fast growing non-pathogenic Kinetoplastida Leishmania tarentolae. The feature that sets Kinetoplastida apart form other eukaryotes is the ability to translate messenger RNA generated by RNA polymerase I or even a foreign polymerase due to natural uncoupling of transcription from RNA processing. In the most successfui implementation the heterologous gene is delivered on a circular episome into the L.tarentoale strain. Although this System was shown to deliver up to 300mg of recombinant protein per liter it suffers from strong clonal variations. Initial results suggests that this may be linked to the behavior of episomal elements in Leishmania cells. Understanding of structural dynamics, inheritance and transcription of the circular and linear episomes information should be essential for constructing new generations of expression vectors and for developing strategies in suppressing episome carrying drug resistance genes in pathogenic strains. In a complementary approach, we plan to develop a high-yield in vitro translation System based on extracts of Ltarentolae. Such a System is expected to be more robust and much less expensive tJian other in vitro translation Systems.