A magnetic bead-based fluorescent substrate for sensitive assay of SARS-CoV-2 3C-like protease activity

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-09-05 DOI:10.1016/j.pep.2025.106811

Thanh-Hoa T. Tran , Trung-Duc Nguyen , Ngoc-Nam Phan , Hang T. Ngo , Phuc-Loc Nguyen Do , Phan-Anh Le , Nho-Thai Dinh , Tuan-Nghia Phan , Hong-Loan T. Nguyen

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引用次数: 0

Abstract

The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6×His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (y = 725.29x − 52.356; R² = 0.998) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a K_m of 22.01 ± 3.5 μM, k_cat of 0.021 s^-¹, and catalytic efficiency (k_cat/K_m) of 946 M^-¹^.s^-¹. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC₅₀ of 0.88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.

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基于磁珠的荧光底物用于sars - cov - 23c样蛋白酶活性的灵敏测定。

SARS-CoV-2的3c样蛋白酶（3CLpro）在病毒多蛋白加工过程中发挥重要作用，是抗病毒药物的重要靶点。在本研究中，我们设计并制作了模块化荧光重组底物（6×His-ECFP-AVLQSGFRK-EYFP），然后将其固定在Ni-NTA磁珠（Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP）上，用于测定3CLpro的活性。在特定的AVLQ↓SG基序上切割后，EYFP片段被释放到上清中，并通过荧光测量（Ex/Em = 480/528 nm）进行定量。得到标准曲线（y = 725.29x - 52.356; R2 = 0.998），可准确定量裂解产物及动力学参数。实验结果表明，该底物的Km为22.01±3.5 μM， kcat为0.021 s-1，催化效率（kcat/Km）为946 m -1 s-1。与SDS-PAGE相比，该方法的灵敏度提高了约50倍，并且还确定了GC376对3CLpro的抑制作用，IC50为0.88 μM。由于模块化底物设计允许替换n端结构域和切割基序，因此我们的底物开发和检测可以扩展到其他高特异性蛋白酶。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.