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 使用体外表达系统产生生物化合物的一个有吸引力的特点是 能够以各种规模生产各种化合物（蛋白质、肽、抗体） 廉价且快速的方式。目前分析这些产生的化合物的方法需要 纯化对于使用表达系统衍生的产品来说是一个主要缺点。 例如，中等丰度（可溶性）蛋白质可能需要四天内 27 个单独的步骤才能获得 净化;筛选单一蛋白质需要花费大量的时间和费用。筛选潜力库 具有治疗价值的生物化合物，在使用之前需要对每种化合物进行许多纯化步骤 可以进行任何分析。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，我们开发了一种独特的基于 DNA 的方案，使用 差示扫描量热法测定纯化的生物化合物的质量 和混合媒体。独特的是，我们的探针普遍适用于多种生物化合物 能够在纯化之前进行质量定量。这一进展允许尽早对生物制剂进行初步分析 在生产过程中，降低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市场上依赖于的众多质量分析套件 比色法或荧光法。这些方法对于化合物类型具有高度特异性 进行了分析，并且存在妨碍普遍适用性的局限性。我们的产品具有卓越的优势 现有方法。在这个项目中，我们计划筛选生物表达的 SARS-CoV-2 刺突文库 和我们的商业合作者产生的核衣壳蛋白。这项工作的目的是评估 我们的测定是否能够仅从质量预测其表达系统的蛋白质产量 测定其未纯化的样品。此外，我们将使用我们的检测来验证蛋白质 纯化前后的结构。