Engineered Pan-Specific Reagents for N-Glycan Detection and Enrichment

用于 N-聚糖检测和富集的工程泛特异性试剂

• 批准号: 10761111
• 负责人: Sheng-Cheng Wu
• 金额: $ 30.17万
• 依托单位: GLYCOSENSORS AND DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761111
• 关键词: Address; Affinity; Affinity Chromatography; Antibodies; Antibody Response; Antigens; Asparagine; Bacteria; Binding; Binding Proteins; Biological; Biological Assay; Biological Markers; Biological Process; Carbohydrates; Cell Extracts; Cells; Complex; Computer Models; Detection; Development; Diagnostic Reagent Kits; Directed Molecular Evolution; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Escherichia coli; Expression Library; F Box Domain; FBXO2 gene; Family; Flow Cytometry; Glycopeptides; Glycoproteins; Immunohistochemistry; Infection; Lectin; Legal patent; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mus; Mutate; Nature; Neurodegenerative Disorders; Peptides; Performance; Phase; Plant Lectins; Plant Resins; Play; Polysaccharides; Positioning Attribute; Procedures; Production; Property; Protein Family; Protein Subunits; Proteins; Protocols documentation; Reagent; Recombinants; Research; Role; Sampling; Serum; Site; Specificity; Structural Models; Structure; Techniques; Technology; Variant; Western Blotting; Yeasts; advanced analytics; carbohydrate binding protein; carbohydrate receptor; design; glycoproteomics; glycosylation; improved; instrumentation; manufacturing scale-up; novel; preference; scale up; screening; sugar; targeted biomarker; tool; ubiquitin ligase; ubiquitin-protein ligase

PROJECT SUMMARY Glycans play crucial roles in nearly every aspect of biological processes, and their distinct properties make them appealing as disease biomarker targets. However, due to their highly branched and variably linked nature, glycans pose a challenge for detection, purification, and structural analysis. Although advanced analytical techniques and instrumentation have been developed that target glycans, there is still a great need for reagents with well-defined carbohydrate specificity and high affinity that can be used to independently interrogate or enrich carbohydrate samples prior to traditional analyses. Lectenz Bio has been engineering glycan-processing enzymes and glycan-binding proteins into high-affinity glycan-specific reagents with tunable properties. The Fbs1 component of the E3 ubiquitin ligase complex recognizes glycans as part of its biological role in ubiquitylation. It is therefore a promising protein for conversion into a binding reagent for asparagine-linked glycans (N-glycans), which we will achieve by employing computational modeling and directed evolution. Initial studies demonstrate that Fbs1 can be readily expressed in E. coli, and binds specifically to N-glycosylated peptides and proteins, and not to the corresponding non-glycosylated peptides and proteins. However, it does demonstrate a preference for certain N-glycan structures. A pan-specific N-glycan binding reagent would directly address the urgent need to tools to help detect glyco- biomarkers and could immediately benefit the field of glycomics/glycoproteomics by enabling sample pre- enrichment. After engineering and optimizing the production of recombinant Fbs1 and its engineered variants, Lectenz Bio will develop a suite of kits for its application in Western blotting, immunohistochemistry, ELISA, flow cytometry, and affinity chromatography.

项目概要 聚糖在生物过程的几乎每个方面都发挥着至关重要的作用，其独特的特性使得 它们作为疾病生物标志物目标很有吸引力。然而，由于它们的高度分支和可变连接 在自然界中，聚糖对检测、纯化和结构分析提出了挑战。虽然先进 针对聚糖的分析技术和仪器已经开发出来，但仍然有很大的需求 适用于具有明确碳水化合物特异性和高亲和力的试剂，可独立用于 在传统分析之前询问或富集碳水化合物样品。 Lectenz Bio 一直致力于将聚糖加工酶和聚糖结合蛋白改造为高亲和力 具有可调特性的聚糖特异性试剂。 E3 泛素连接酶复合物的 Fbs1 成分 识别聚糖作为其泛素化生物学作用的一部分。因此，它是一种很有前途的蛋白质 转化为天冬酰胺连接聚糖（N-聚糖）的结合试剂，我们将通过以下方式实现 采用计算建模和定向进化。初步研究表明 Fbs1 可以很容易地 在大肠杆菌中表达，并特异性结合 N-糖基化肽和蛋白质，而不是结合 相应的非糖基化肽和蛋白质。然而，它确实表现出对某些特定的偏好 N-聚糖结构。 泛特异性 N-聚糖结合试剂将直接解决对帮助检测糖的工具的迫切需求。 生物标志物，并且可以通过使样品预制备立即使糖组学/糖蛋白质组学领域受益 丰富。经过工程设计和优化重组 Fbs1 及其工程变体的生产后， Lectenz Bio 将开发一套用于蛋白质印迹、免疫组织化学、ELISA、 流式细胞术和亲和层析。