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.

摘要 使用体外表达系统产生生物化合物的一个吸引人的特征是 能够在不同的规模上生产各种化合物(蛋白质、多肽、抗体)，在 廉价而快捷的方式。目前分析这些生成的化合物的方法都需要 纯化，这是使用来自表达系统的产品的主要缺点。 例如，一种中等含量的(可溶)蛋白质可能需要在四天内进行27个单独的步骤才能 纯化：筛选一种单一蛋白质需要大量的时间和费用。筛选有潜力的文库 具有治疗价值的生物化合物，在此之前需要对每种化合物进行许多纯化步骤 任何分析都可以执行。这包括简单的测量，就像确定 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两种纯品中生物化合物的质量 和混合媒体。独一无二的是，我们的探测器普遍适用于各种生物化合物。 能够在提纯之前进行质量量化。这一进展使生物制品的初步分析得以及早进行。 在生产过程中，降低相关的时间要求和资源成本。一个额外的好处 是否有可能取代目前市场上大量依赖于 比色法或荧光法。这些方法对化合物的类型有很高的特异性 进行了测试，但存在妨碍普遍适用的局限性。我们的产品具有优越的优势。 现有的方法。在这个项目中，我们计划筛选一个生物表达的SARS-CoV-2尖峰蛋白文库 以及由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 我们的检测方法是否能够仅根据质量预测它们的表达系统的蛋白质产量 对其未净化样品的检测。此外，我们将使用我们的化验来验证蛋白质 结构在提纯前后。