Humanizing N-linked glycosylation in Escherichia coli

描述（由申请人提供）：基于蛋白质的治疗药物目前占FDA批准的新药的四分之一，市场份额超过300亿美元。绝大多数治疗性蛋白需要额外的翻译后修饰才能实现其完整的生物学和治疗功能。其中最重要的修饰之一，n -连接蛋白糖基化，预计会影响一半以上的真核蛋白物种，并且对于大量人类治疗性蛋白的正确折叠、药代动力学稳定性、系统半衰期、免疫原性和整体功效通常是必不可少的。由于大多数细菌不会将自身的蛋白质糖基化，因此许多与治疗相关的糖蛋白（包括抗体）的表达被转移到哺乳动物细胞中。不幸的是，哺乳动物细胞培养存在许多缺点，包括体积生产力低、产品异质性、培养基成本高、逆转录病毒污染以及产生稳定细胞系所需的时间相对较长。然而，最近有报道称，空肠弯曲杆菌（Campylobacter jejuni）天冬酰胺连接（Nlinked）蛋白糖基化系统可以功能性地转移到大肠杆菌中，使受体大肠杆菌细胞具有糖化蛋白的能力。虽然细菌的n -聚糖在结构上与真核生物的n -聚糖不同，但有理由认为，这种使糖激活的大肠杆菌可以被改造成模仿人类和其他高等哺乳动物早期处理n -聚糖的顺序糖基化反应。这一成就将为在细菌中进行复杂的类人蛋白糖基化打开大门。因此，本研究项目的长期目标是“人源化”大肠杆菌中n -连接蛋白糖基化过程，以便在这种简单的宿主生物中常规生产真正的人糖蛋白。为了实现这一目标，这一特殊应用的目的是通过聚糖工程（特异性目标1）和将新聚糖位点特异性转移到靶蛋白上（特异性目标2），开始“人源化”细菌糖基化系统的早期阶段。提出的研究背后的具体假设是，大肠杆菌中真核n-糖基化途径的重建将导致n-糖蛋白具有结构均匀的类人聚糖。这些研究是重要的，因为它们应该(i)提供必要的遗传工具来帮助阐明糖基化在无数生物现象中的关键作用，（ii）使新的糖缀合物和潜在的免疫刺激剂的生物技术合成能够用于研究，工业和治疗应用。

DESCRIPTION (provided by applicant): Protein-based therapeutics currently represent one in every four new drugs approved by the FDA and command a market in excess of $30 billion. The vast majority of therapeutic proteins require additional post-translational modifications to attain their full biological and therapeutic function. One of the most important of these modifications, N-linked protein glycosylation, is predicted to affect more than half of all eukaryotic protein species and is often essential for proper folding, pharmacokinetic stability, systemic half-life, immunogenicity and overall efficacy for a large number of human therapeutic proteins. Since most bacteria do not glycosylate their own proteins, expression of many therapeutically relevant glycoproteins, including antibodies, is relegated to mammalian cells. Unfortunately, mammalian cell culture suffers from a number of drawbacks including low volumetric productivity, product heterogeneity, high media cost, retroviral contamination and the relatively long time required to generate stable cell lines. Recently, however, it has been reported that the Campylobacter jejuni asparagine-linked (Nlinked) protein glycosylation system can be functionally transferred into Escherichia coli, giving the recipient E. coli cells the ability to glycosylate proteins. Although the bacterial N-glycan is structurally different from its eukaryotic counterparts, it stands to reason that such glyco-enabled E. coli could be engineered to perform sequential glycosylation reactions that mimic the early processing of N-glycans in humans and other higher mammals. Such an accomplishment would open the door for performing complex human-like protein glycosylation in bacteria. Thus, the long-term goal of this research project is to "humanize" the N-linked protein glycosylation process in E. coli for the routine production of authentic human glycoproteins in this simple host organism. Towards this goal, the objective of this particular application is to begin the early stages of "humanizing" the bacterial glycosylation system via glycan engineering (specific aim 1) and site-specific transfer of novel glycans onto target proteins (specific aim 2). The specific hypothesis behind the proposed research is that reconstitution of a eukaryotic N-glycosylation pathway in E. coli will result in N-glycoproteins with structurally homogeneous human-like glycans. These studies are significant because they should (i) provide the necessary genetic tools to help elucidate the crucial role of glycosylation in a myriad of biological phenomena and (ii) enable the biotechnological synthesis of novel glycoconjugates and potential immunostimulating agents for research, industrial and therapeutic applications. PUBLIC HEALTH RELEVANCE: N-linked glycosylation is predicted to affect more than half of all eukaryotic protein species and is often essential for proper folding, pharmacokinetic stability, systemic half-life, immunogenicity and overall efficacy for a large number of therapeutically relevant proteins (e.g., human erythropoietin). Since most bacteria do not glycosylate their own proteins, expression of many therapeutically relevant glycoproteins, including antibodies, is relegated to mammalian cells despite the fact that mammalian cell culture suffers from a number of well-documented drawbacks. Therefore, the focus of these studies is the development of "glyco-engineered" bacteria that are capable of producing authentic human glycoproteins.