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.

大多数药品合成的中间步骤，包括治疗蛋白，基因组支持的个性化医学和抗病毒疫苗，是从复杂的生物混合物中分离出感兴趣的靶化合物。 感兴趣的物种的分离是一个复杂的资源密集型多步骤过程，但对于确保所得制药产品的纯度和质量至关重要。药物通常通过色谱分离，其中复杂的混合物被多个装有固体树脂颗粒的床传递。 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穿过床，如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相互作用所决定的。 该项目将开发新的色谱树脂，通过将化学配体授予与感兴趣的化合物具有结合相互作用的固体，从而更有效地将生物混合物分离，从而选择性地阻碍了其通过包装柱的运动。 可以预料，这种多模式配体，即与特定化合物有多种相互作用的配体，将显着降低生物混合物色谱纯化的复杂性，因此成本。 具体而言，该项目将在基因上设计酶的活性位点，从而使酶将设计器配体的特性合成，从而使它们更具选择性地与复杂混合物的靶向蛋白结合。 该项目中要开发的结合位点是基于治疗性蛋白质特异性聚糖的配体（或糖原），该配体将通过一种新型的生物合成方法合成，将其合成。 这些酶比传统方法具有多个优点，包括可以轻松地设计它们以量身定制反应特异性，以产生高度生物相容性的配体以进行蛋白质纯化。这项工作的核心假设是多功能性糖合酶酶的遗传工程将导致这些酶对良好量身定制的糖体的能力，而糖化剂反过来又将具有基于多峰配体蛋白质色谱的理想选择性。将设计一个酶库，以生成活性和选择性结合的配体树脂库。最终的多模式相互作用（包括识别，结合和解开）将使用高吞吐量实验平台进行表征。使用各种糖酮和aglycone部分合成的糖化体具有微调的氢键，疏水和/或静电相互作用，将促进具有定义的表面特性的靶向纯化蛋白质。这项工作将导致新的生物材料，实验工具和定量结构 - 特性关系模型的发展，以预测蛋白质 - 聚糖相互作用，这些蛋白质 - 聚糖相互作用将推动从糖生物学到生物释放的各种领域。本科生和研究生将在蛋白质工程和生物序列的跨学科领域进行培训，研究的概念将被纳入本科课程，并将其纳入罗格斯大学的外展/培训计划。 PI将与大学和工业合作伙伴合作，测试设计师Glycoligands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.

Carbohydrate-Active enZyme (CAZyme) enabled glycoengineering for a sweeter future

• DOI: 10.1016/j.copbio.2020.09.006
• 发表时间: 2020-12-01
• 期刊: CURRENT OPINION IN BIOTECHNOLOGY
• 影响因子: 7.7
• 作者: Bandi, Chandra Kanth;Agrawal, Ayushi;Chundawat, Shishir P. S.
• 通讯作者: Chundawat, Shishir P. S.

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.