Engineering Escherichia coli for glycosylation of complex human proteins

改造大肠杆菌以糖基化复杂的人类蛋白质

• 批准号: 8332786
• 负责人: Adam Charles Fisher
• 金额: $ 71.49万
• 依托单位: GLYCOBIA, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8332786
• 关键词: Accounting; Affect; Anabolism; Archaea; Asparagine; Bacteria; Benchmarking; Biotechnology; Capital; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Chinese Hamster Ovary Cell; Collection; Communities; Complex; Culture Media; Cytidine Monophosphate N-Acetylneuraminic Acid; Development; Drug Kinetics; Engineering; Enzymes; Escherichia coli; Feedback; Galactosyltransferases; Genes; Glycoproteins; Goals; Gram-Negative Bacteria; Growth; Half-Life; Healthcare; Heterogeneity; Human; Insecta; Interferons; Laboratories; Lead; Link; Mammalian Cell; Organism; Outcome; Pathway interactions; Patients; Pharmacologic Substance; Phase; Plague; Plants; Polysaccharides; Post-Translational Protein Processing; Predisposition; Price; Process; Production; Productivity; Protein Glycosylation; Proteins; Protozoa; Recombinant Proteins; Research; Screening procedure; Sialyltransferases; Site; Solutions; Somatropin; Structure; System; Therapeutic; Time; TimeLine; Transfection; Variant; Viral; Yeasts; anakinra; antibody engineering; base; clinical efficacy; cost effective; dolichyl-diphosphooligosaccharide - protein glycotransferase; epimerase; glycosylation; glycosyltransferase; improved; in vivo; large scale production; link protein; manufacturing facility; phase 1 study; programs; stable cell line; success; sugar; therapeutic protein; therapeutic target

DESCRIPTION (provided by applicant): Escherichia coli was the host organism for production of the first approved recombinant protein therapeutic in 1982. We now know that most therapeutic proteins require N-linked protein glycosylation to achieve their full clinical efficacy. Since E. coli has not been capable of protein glycosylation, the majority of approved therapeutic proteins are now expressed in mammalian host cells. While mammalian cells can express N-linked glycoproteins, they can have several drawbacks including: (i) slow growth, (ii) expensive media, (iii) long development timelines, (iv) low volumetric productivity, (v) susceptibility to viral contamination, and (vi) product heterogeneity. This problem has not gone unnoticed by the scientific community, and several eukaryotic organisms have been re-engineered for expression of therapeutic glycoproteins. Unfortunately, all eukaryotic hosts - including Chinese hamster ovary cells, plant cells, insect cells, or even genetically engineered yeast - introduce nonhuman glycoforms that arise from native glycosylation pathways. Glycobia specializes in glycoengineering bacteria as a platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis of these proposed studies is that glycoengineered E. coli can be used to express therapeutic glycoproteins. In Phase I of this project, we engineered E. coli capable of glycosylating proteins with the eukaryotic core glycan (Man3GlcNAc2) that is the predominant glycan in both plant and insect cells. In Phase II of this project, we propose to further engineer E. coli to enable glycosylation of therapeutic proteins with terminally sialylated human glycans. Specifically, we propose to engineer E. coli to glycosylate therapeutic proteins with eukaryotic N-glycans by screening enzymes to: (i) preferentially glycosylate N-X-S/T glycosylation motifs and (ii) efficiently glycosylate therapeutic target proteins with eukaryotic glycans. Further, we propose to engineer E. coli to synthesize and transfer complex terminally sialylated N-glycans by: (i) extending the Man3GlcNAc2 biosynthetic pathway for the biosynthesis of terminally sialylated glycans and (ii) screening enzymes for their ability to transfer the complex human N-glycan to target proteins. The benchmark of success for this project is expression of a commercial glycoprotein in E. coli. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoproteins for both companies and patients.

描述（由申请人提供）：大肠杆菌是1982年第一个被批准的重组蛋白治疗药物的宿主生物。我们现在知道，大多数治疗性蛋白需要n -连接蛋白糖基化才能达到充分的临床疗效。由于大肠杆菌不具备蛋白糖基化的能力，大多数被批准的治疗性蛋白现在都在哺乳动物宿主细胞中表达。虽然哺乳动物细胞可以表达n -连接糖蛋白，但它们可能有几个缺点，包括：(i)生长缓慢，（ii）培养基昂贵，（iii）发育时间长，（iv）体积生产力低，(v)易受病毒污染，（vi）产品异质性。这个问题并没有被科学界忽视，一些真核生物已经被重新设计用于表达治疗性糖蛋白。不幸的是，所有真核宿主——包括中国仓鼠卵巢细胞、植物细胞、昆虫细胞，甚至是基因工程酵母——都会引入由天然糖基化途径产生的非人类糖型。glybia专门从事糖工程细菌作为立体特异性生物合成治疗糖蛋白的平台。这些研究的具体假设是糖工程大肠杆菌可以用来表达治疗性糖蛋白。在该项目的第一阶段，我们设计了能够将蛋白质与真核核心聚糖（Man3GlcNAc2）糖基化的大肠杆菌，该聚糖是植物和昆虫细胞中的主要聚糖。在这个项目的第二期，我们建议进一步改造大肠杆菌，使治疗蛋白的糖基化与最终唾液化的人聚糖。具体来说，我们建议通过筛选酶来改造大肠杆菌，使其与真核n -聚糖结合使治疗蛋白糖基化：(i)优先使N-X-S/T糖基化基序糖基化；（ii）有效地使治疗靶蛋白与真核聚糖糖基化。此外，我们建议设计大肠杆菌来合成和转移复杂的末端唾液化n -聚糖，方法是：(i)扩展Man3GlcNAc2生物合成途径，用于末端唾液化聚糖的生物合成；（ii）筛选能够将复杂的人n -聚糖转移到目标蛋白的酶。这个项目成功的基准是在大肠杆菌中表达了一种商业糖蛋白。这种细菌表达平台代表了一种革命性的解决方案，解决了为公司和患者生产具有成本效益的糖蛋白这一尚未解决的生物医学挑战。