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

基本信息

批准号：
8203830
负责人：
Adam Charles Fisher
金额：
$ 88.38万
依托单位：
GLYCOBIA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8203830
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

项目摘要

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. PUBLIC HEALTH RELEVANCE: Most approved therapeutic proteins require posttranslational N-linked protein glycosylation and, as a consequence, are expressed in eukaryotic host cells that can be expensive, susceptible to viral contamination, and prone to product heterogeneity. The outcomes are low profit margins for biotechnology and pharmaceutical companies and prices that are prohibitive to the healthcare consumer. The proposed studies focus on expressing safe, affordable, and controlled complex human glycoproteins in the simple bacterium Escherichia coli.

描述（由申请人提供）：大肠杆菌是 1982 年第一个批准的重组蛋白治疗剂生产的宿主生物。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实现其完整的临床功效。由于大肠杆菌无法进行蛋白质糖基化，因此大多数已批准的治疗性蛋白质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) 昂贵的培养基，(iii) 开发时间长，(iv) 体积生产率低，(v) 易受病毒污染，以及（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。 Glycobia 专门研究糖工程细菌作为治疗性糖蛋白立体特异性生物合成的平台。这些拟议研究的具体假设是糖工程大肠杆菌可用于表达治疗性糖蛋白。在该项目的第一阶段，我们设计了能够用真核核心聚糖 (Man3GlcNAc2) 糖基化蛋白质的大肠杆菌，真核核心聚糖是植物和昆虫细胞中的主要聚糖。在该项目的第二阶段，我们建议进一步改造大肠杆菌，以实现治疗性蛋白质与末端唾液酸化的人类聚糖的糖基化。具体来说，我们建议通过筛选酶来改造大肠杆菌，使其用真核 N-聚糖糖基化治疗性蛋白质：(i) 优先糖基化 N-X-S/T 糖基化基序，以及 (ii) 用真核聚糖有效糖基化治疗靶蛋白。此外，我们建议通过以下方式改造大肠杆菌来合成和转移复杂的末端唾液酸化的N-聚糖：（i）扩展Man3GlcNAc2生物合成途径以生物合成末端唾液酸化的聚糖，以及（ii）筛选酶的转移复杂的人类聚糖的能力N-聚糖靶向蛋白质。该项目成功的基准是商业糖蛋白在大肠杆菌中的表达。该细菌表达平台代表了一种变革性的解决方案，以应对尚未解决的生物医学挑战，即为公司和患者生成具有成本效益的糖蛋白。公共健康相关性：大多数批准的治疗性蛋白需要翻译后 N 连接蛋白糖基化，因此在真核宿主细胞中表达，而真核宿主细胞可能价格昂贵、容易受到病毒污染，并且容易产生产品异质性。其结果是生物技术和制药公司的利润率很低，而医疗保健消费者的价格却令人望而却步。拟议的研究重点是在简单细菌大肠杆菌中表达安全、负担得起且受控的复杂人类糖蛋白。