SusChEM: Designer Glycoligands for Enabling Targeted Multimodal Protein Bioseparations

SusChEM：用于实现靶向多模式蛋白质生物分离的设计糖配体

• 批准号: 1704679
• 负责人: Shishir Chundawat
• 金额: $ 30万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1704679&HistoricalAwards=false
• 关键词: SusChEM; Designer; Glycoligands; Enabling; Targeted

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.

大多数药物产品（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）合成的中间步骤是从复杂的生物混合物中分离目标化合物。 目标物质的分离是一个复杂的、资源密集型的多步骤过程，但对于确保所得药物产品的纯度和质量至关重要。药物通常通过色谱法分离，其中复杂的混合物通过填充有固体树脂颗粒的多个床。 虽然固体通常是惰性的，但混合物的不同组分以不同的速度通过床，这取决于混合物中存在的每个分子的大小、其行进路径以及与可能阻碍运动的固体表面的相互作用。 该项目将开发新的色谱树脂，更有效地分离生物混合物，通过赋予化学配体的固体，具有与感兴趣的化合物，选择性地阻止它们通过填充柱的运动结合相互作用。 预期这种多峰配体，即与特定化合物具有多种相互作用的那些，将显著降低生物混合物的色谱纯化的复杂性，并因此降低其成本。 具体来说，该项目将对酶的活性位点进行基因工程改造，使酶能够合成具有特定特性的设计配体，使它们更有选择性地与复杂混合物中的靶蛋白结合。 在这个项目中开发的结合位点是治疗性蛋白质特异性聚糖基配体（或糖配体），其将通过融合工程化糖合酶的新型生物合成方法合成。 与传统方法相比，这些酶提供了几个优点，包括它们可以容易地被工程化以定制反应特异性，从而产生用于蛋白质纯化的高度生物相容性配体。这项工作的中心假设是，多功能糖合酶的基因工程将导致这些酶的能力，以精细定制糖配体，这反过来，将具有理想的选择性多模式的配体为基础的蛋白质色谱。将设计酶库以产生活性和选择性结合配体树脂库。由此产生的多模态相互作用，包括识别，结合和解结合，将使用高通量实验平台进行表征。使用具有微调氢键、疏水和/或静电相互作用的各种糖基和糖苷配基部分合成的糖配体将促进具有限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，实验工具和定量结构-性质关系模型，用于预测蛋白质-聚糖相互作用，这将推动从糖生物学到生物分离的各个领域。本科生和研究生将在蛋白质工程和生物分离的跨学科领域进行培训，研究的概念将被纳入罗格斯大学的本科课程和外展/培训计划。PI将与大学和工业合作伙伴合作，测试设计师糖配体的商业潜力。

项目成果

Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

• DOI: 10.1101/2020.03.23.001982
• 发表时间: 2020-03
• 期刊: bioRxiv
• 作者: Ayushi Agrawal;C. K. Bandi;Tucker Burgin;Y. Woo;H. Mayes;Shishir P. S. Chundawat

Engineered Regulon to Enable Autonomous Azide Ion Biosensing, Recombinant Protein Production, and in Vivo Glycoengineering

工程调节子可实现自主叠氮离子生物传感、重组蛋白生产和体内糖工程

• DOI: 10.1021/acssynbio.0c00449
• 发表时间: 2021
• 期刊: ACS Synthetic Biology
• 影响因子: 4.7
• 作者: Bandi, Chandra Kanth;Skalenko, Kyle S.;Agrawal, Ayushi;Sivaneri, Neelan;Thiry, Margaux;Chundawat, Shishir P.
• 通讯作者: Chundawat, Shishir P.

Synthetic promoter based azide biosensor toolkit to advance chemical-biology

基于合成启动子的叠氮化物生物传感器工具包，以推进化学生物学

• DOI: 10.1101/2020.07.08.193060
• 发表时间: 2020
• 期刊: bioRxiv
• 作者: Bandi, C. K.;Skalenko, K. S.;Agrawal, A.;Sivaneri, N.;Thiry, M.;Chundawat, S. P.
• 通讯作者: Chundawat, S. P.