Non-pathogenic trypanosomatid protozoa as a platform for protein research and production
Non-pathogenic trypanosomatid protozoa as a platform for protein research and production
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DOI：
10.1016/s1046-5928(02)00001-3
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发表时间：
2002-07-01
影响因子：
1.6
通讯作者：
Alexandrov, K
中科院分区：
文献类型：
--
作者：
Breitling, R;Klingner, S;Alexandrov, K
目前存在的所有真核蛋白表达系统都是基于自主生命形式。为了利用与寄生生物体相关的潜在实际益处，我们已经开发了一种基于蜥蜴的原生动物寄生虫利什曼原虫（锥虫科）的新蛋白表达系统。为了实现强转录，将感兴趣的基因整合到小亚基核糖体RNA基因中。获得的表达水平高达每升悬浮培养物30 mg的重组蛋白，并且随着整合的基因拷贝数线性增加。为了评估该系统生产后修饰蛋白质的潜力，我们在L。tarentolae从培养上清液中分离的重组蛋白具有生物活性，在N-末端天然加工，并且N-糖基化。N-糖基化异常均匀，具有双触角型寡糖和Man（3）GlcNAc（2）核心结构，占存在的聚糖的90%以上。L.因此，tarentolae是第一个描述的生物技术上有用的单细胞真核生物，其产生双触角完全半乳糖基化的核心-α-1，6-岩藻糖基化的N-聚糖。(C)2002 Elsevier Science（美国）。All rights reserved.
All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system's potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogenous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-alpha-1,6-fucosylated N-glycans. (C) 2002 Elsevier Science (USA). All rights reserved.