DNA-based assay for calorimetric determination and quantitation of protein concentrations in pure or mixed solutions

基于 DNA 的测定法，用于纯溶液或混合溶液中蛋白质浓度的量热测定和定量

• 批准号: 10547595
• 负责人: Matthew Walter Eskew
• 金额: $ 25.86万
• 依托单位: THERMOCAP LABORATORIES INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-06 至 2024-01-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547595
• 关键词: 2019-nCoV; ACE2; Address; Antibodies; Biological; Biological Assay; Biological Products; Buffers; Characteristics; Clinical Trials; Collaborations; Complex; Complex Mixtures; Consumption; Culture Media; DNA; Diagnostic Reagent Kits; Differential Scanning Calorimetry; Engineering; Environment; Equipment; Feedback; Fluorescence; Glass; In Vitro; Individual; Instruction; Laboratories; Libraries; Mass Spectrum Analysis; Measurement; Methods; Modification; Molecular Target; Nucleocapsid Proteins; Outcome; Pain; Peptide antibodies; Peptides; Pharmaceutical Preparations; Phase; Process; Production; Property; Protein Analysis; Proteins; Reagent; Resources; Sampling; Scanning; Scheme; Surveys; System; Techniques; Testing; Therapeutic; Thermodynamics; Time; Vial device; Viral Proteins; Work; base; cost; data reduction; design; experience; instrument; melting; novel; novel therapeutics; prevent; protein structure; response; screening; validation studies

Abstract An attractive feature of using in vitro expression systems to generate biological compounds is the capability to produce a wide variety of compounds (proteins, peptides, antibodies) on various scales, in a cheap and rapid manner. Current methods to analyze any of these produced compounds require purification which poses a major drawback for working with products derived from expression systems. For example, a moderately abundant (soluble) protein can require 27 individual steps over four days to purify; a significant amount of time and expense to screen a single protein. Screening a library of potential biological compounds for therapeutic value, requires many purification steps for each compound before any analysis can be performed. This includes measurements as simple as determination of the mass of the produced biological compounds. To this end we have developed a unique DNA-based scheme using differential scanning calorimetry for determination of the masses of biological compounds in both purified and mixed media. Uniquely, our probes are universally applicable to a wide range of biological compounds enabling mass quantification prior to purification. This advance allows for initial analysis of biologics early in the production process, lowering associated time requirements and resource costs. An added benefit is the possibility to supplant the numerous mass analysis kits currently on the market that rely on colorimetric or fluorescent methods. These methods are highly specific to the types of compounds assayed and have limitations preventing universal applicability. Our product offers a superior advantage of existing methods. In this project we plan to screen a library of biologically expressed SARS-CoV-2 spike and nucleocapsid proteins generated by our commercial collaborator. The aim of this work is to evaluate whether our assay is capable of predicting protein yields from their expression systems solely from a mass determination of their unpurified samples. Additionally, we will use our assay to verify the protein structure before and after purification.

抽象的 使用体外表达系统生成生物化合物的一个吸引人特征是 在各种尺度上产生多种化合物（蛋白质，肽，抗体）的能力 便宜而快速的方式。当前分析这些产生化合物中的任何方法都需要 纯化为使用来自表达系统的产品而构成主要缺点。 例如，中等丰富的（可溶）蛋白可以在四天内需要27个单独的步骤 净化;筛选单个蛋白质的大量时间和费用。筛选潜力的库 用于治疗价值的生物化合物，需要每种化合物的纯化步骤 任何分析都可以进行。这包括像确定质量一样简单的测量 产生的生物化合物。为此，我们使用 差异扫描量热法，以测定两者纯化的生物化合物质量 和混合媒体。独特的探针普遍适用于广泛的生物化合物 在纯化之前实现质量定量。这种进步允许早期对生物制剂进行初步分析 在生产过程中，降低了相关的时间要求和资源成本。额外的好处 是否有可能取代目前依赖市场上的众多群众分析工具包 比色或荧光方法。这些方法是高度特异性的。 测定并具有阻止普遍适用性的局限性。我们的产品提供了优势 现有方法。在这个项目中，我们计划筛选一个生物表达的SARS-COV-2 Spike库 和我们的商业合作者产生的核素蛋白。这项工作的目的是评估 我们的测定是否能够仅从质量中预测其表达系统的蛋白质产量 确定其未纯净的样品。此外，我们将使用我们的测定来验证蛋白质 纯化前后的结构。