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

摘要

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.