SusChEM: Designer Glycoligands for Enabling Targeted Multimodal Protein Bioseparations

在大多数药物产品的合成中，包括治疗性蛋白质、基因组使能的个性化药物和抗病毒疫苗，中间步骤是从复杂的生物混合物中分离感兴趣的目标化合物。分离感兴趣的物种是一个复杂的、资源密集的多步骤过程，但对于确保所产生的药物产品的纯度和质量是必不可少的。药物通常通过层析分离，在层析中，复杂的混合物通过装满固体树脂颗粒的多个床。尽管固体通常是惰性的，但混合物的不同成分以不同的速度通过床层，这取决于混合物中存在的每个分子的大小、其传播路径以及可能阻碍运动的与固体表面的相互作用。该项目将开发新的色谱树脂，通过向固体赋予化学配体，这些配体与感兴趣的化合物具有结合作用，从而选择性地阻碍它们在填充柱中的运动，从而更有效地分离生物混合物。预计这种多模式配体，即那些与特定化合物具有多种相互作用的配体，将显著降低生物混合物的色谱纯化的复杂性，从而降低成本。具体地说，该项目将对酶的活性部位进行基因工程，以便酶将合成具有使其更有选择性地与复杂混合物中的目标蛋白质结合的属性的设计者配体。该项目将开发的结合位点是治疗性蛋白质特异性的基于糖链的配体(或糖配体)，将通过融合工程糖合酶酶的新的生物合成方法来合成。与传统的方法相比，这些酶有几个优点，包括它们可以很容易地进行工程设计，以调整反应的特异性，从而产生用于蛋白质纯化的高度生物相容性的配体。这项工作的中心假设是多功能糖合成酶的基因工程将导致这些酶具有精细定制糖配体的能力，这反过来将对基于多模式配体的蛋白质层析具有理想的选择性。将设计一个酶库来产生一个活性和选择性结合配体树脂库。由此产生的多模式交互，包括识别、绑定和解除绑定，将使用高通量实验平台进行表征。利用具有微调氢键、疏水和/或静电相互作用的不同糖酮和苷元部分合成的糖配体将促进具有特定表面性质的蛋白质的靶向纯化。这项工作将导致开发新的生物材料、实验工具和用于预测蛋白质-多糖相互作用的定量结构-性质关系模型，这将推动从糖生物学到生物分离的多个领域的发展。本科生和研究生将接受蛋白质工程和生物分离跨学科领域的培训，研究中的概念将被纳入本科课程和罗格斯大学的外展/培训计划。PI将与大学和工业合作伙伴合作，测试设计师糖基化的商业潜力。

An intermediate step in the synthesis of most pharmaceutical products, including therapeutic proteins, genome-enabled personalized medicine, and anti-viral vaccines, is the isolation of the target compound of interest from a complex biological mixture. Separation of the species of interest is a complex, resource-intensive multi-step process, but is essential to ensure the purity and quality of the resulting pharmaceutical product. Pharmaceuticals are generally separated via chromatography, in which a complex mixture is passed over multiple beds packed with solid resin particles. Although the solids are generally inert, different components of the mixture pass through the bed at different velocities, as dictated by the size of each molecule present in the mixture, its travel path, and interactions with the solid surface that may impede motion. This project will develop new chromatographic resins that more efficiently separate biological mixtures, by imparting chemical ligands to the solid that have binding interactions with the compounds of interest that selectively impede their motion through the packed column. It is anticipated that such multimodal ligands, i.e. those that have multiple interactions with specific compounds, will significantly reduce the complexity, and thus the cost, of chromatographic purification of biological mixtures. Specifically, this project will genetically engineer the active sites of enzymes, such that the enzymes will synthesize designer ligands with properties that make them more selectively bind to targeted proteins from the complex mixture. The binding sites to be developed in this project are therapeutic protein-specific glycan-based ligands (or glycoligands), which will be synthesized via a novel biosynthetic approach fusing engineered glycosynthase enzymes. These enzymes offer several advantages over traditional approaches, including that they can be easily engineered to tailor the reaction specificity to produce highly biocompatible ligands for protein purification. The central hypothesis of this work is that genetic engineering of multifunctional glycosynthase enzymes will lead to the ability of these enzymes to finely tailor glycoligands, which in turn, will have desirable selectivity for multimodal ligand based protein chromatography. A library of enzymes will be designed to produce a library of active and selective binding ligand resins. The resulting multimodal interactions, including recognition, binding, and unbinding, will be characterized using a high throughput experimental platform. Glycoligands synthesized using various glycone and aglycone moieties with fine-tuned hydrogen bonding, hydrophobic, and/or electrostatic interactions will facilitate targeted purification of proteins with defined surface properties. This work will lead to the development of new biomaterials, experimental tools, and quantitative structure-property relationship models for predicting protein-glycan interactions that will advance diverse fields ranging from glycobiology to bioseparations. Undergraduate and graduate students will be trained in the interdisciplinary areas of protein engineering and bioseparations, and concepts from the research will be incorporated into an undergraduate course and into an outreach/training program at Rutgers University. The PI will partner with university and industrial partners to test the commercial potential of the designer glycoligands.