Kushnir Susanna, Gase Klaus, Breitling Reinhard, Alexandrov Kirill
Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany.
Protein Expr Purif. 2005 Jul;42(1):37-46. doi: 10.1016/j.pep.2005.03.004. Epub 2005 Mar 25.
Production of functional eukaryotic proteins in recombinant form is a bottle-neck in various post-genomic applications and in life science in general. At least partially this is due to the problems associated with the use of endogenous RNA polymerase II for high-level transcription of heterologous genes in eukaryotic expression systems. To circumvent these problems we developed a new inducible protein expression system based on the protozoan host Leishmania tarentolae (Trypanosomatidae). We have created a strain of L. tarentolae constitutively co-expressing T7 RNA polymerase and tetracycline repressor. This strain could be stably transformed with the heterologous target gene under control of the T7 promoter/TET operator assembly, which can initiate transcription upon addition of tetracycline to the culture medium. Using this system, we demonstrated that enhanced green fluorescent protein (EGFP) could be overexpressed to a level of ca. 1% of total cellular protein. The developed system was tested for its ability to inducibly co-express multiple genes. Using two copies of the egfp gene integrated at two different genomic sites, we could obtain expression levels reaching 4% of total cellular protein. Further possible improvements and applications of the developed system are discussed.
以重组形式生产功能性真核蛋白是各种后基因组应用以及整个生命科学中的一个瓶颈。至少部分原因是与在真核表达系统中使用内源性RNA聚合酶II进行异源基因的高水平转录相关的问题。为了规避这些问题，我们基于原生动物宿主大利什曼原虫（锥虫科）开发了一种新的诱导型蛋白表达系统。我们创建了一种组成型共表达T7 RNA聚合酶和四环素阻遏物的大利什曼原虫菌株。该菌株可以用在T7启动子/TET操纵子组件控制下的异源靶基因进行稳定转化，在向培养基中添加四环素后可以启动转录。使用该系统，我们证明增强型绿色荧光蛋白（EGFP）可以过表达至约占总细胞蛋白1%的水平。测试了所开发系统诱导共表达多个基因的能力。使用整合在两个不同基因组位点的两个egfp基因拷贝，我们可以获得达到总细胞蛋白4%的表达水平。讨论了所开发系统进一步可能的改进和应用。
