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

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 以各种规模生产各种化合物（蛋白质、肽、抗体）的能力， 廉价和快速的方式。目前分析这些产生的化合物的方法需要 纯化，这对处理来自表达系统的产物造成了主要的缺点。 例如，中等丰度的（可溶性）蛋白质可能需要在四天内进行27个单独的步骤， 纯化;大量的时间和费用来筛选单一蛋白质。筛选潜在的 对于具有治疗价值的生物化合物，在纯化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。这包括像确定物体质量这样简单的测量。 所产生的生物化合物。为此，我们开发了一种独特的基于DNA的方案， 差示扫描量热法用于测定两种纯化的生物化合物的质量 混合媒体。独特的是，我们的探针普遍适用于广泛的生物化合物 使得能够在纯化之前进行质量定量。这一进展允许早期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。一个额外的好处 有可能取代目前市场上依赖于 比色法或荧光法。这些方法对化合物的类型具有高度特异性 分析，并具有限制，防止普遍适用性。我们的产品具有上级优势 现有的方法。本项目旨在筛选SARS-CoV-2刺突病毒的生物表达文库 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预测蛋白质产量从他们的表达系统仅从质量 测定其未纯化的样品。此外，我们将使用我们的检测方法来验证蛋白质 纯化前后的结构。

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.