改良的双荧光素酶报告（DLR）检测法测定蛋白质稳定性。

Improved dual luciferase reporter (DLR) assay to determine the protein stability.

机构信息

National Key Facility for Crop Gene Resources and Genetic Improvement, ICS, CAAS, 12 Zhongguancun South Street, Haidian District, Beijing, 100081, China.

出版信息

Anal Biochem. 2021 Jan 1;612:114021. doi: 10.1016/j.ab.2020.114021. Epub 2020 Nov 12.

DOI:10.1016/j.ab.2020.114021

PMID:33189704

Abstract

We developed a binary vector co-expressing firefly luciferase (FF) and Renilla luciferase (REN) to detect protein stability in response to different stimuli, and verified the functionality of the vector. The StrigoQuant-like reporter expressing FF and REN in one transcript is a sensitive tool for detecting protein abundance in different genotypes. However, we found that significant differences in the relative FF/REN ratio of empty StrigoQuant vector in different genotypes. Therefore, to determine the actual protein abundance, the relative FF/REN ratio of the protein of interest should be normalized to that of the empty vector.

摘要

我们开发了一种共表达萤火虫荧光素酶 (FF) 和海肾荧光素酶 (REN) 的二元载体，以检测对不同刺激的蛋白质稳定性，并验证了该载体的功能。在一个转录本中表达 FF 和 REN 的 StrigoQuant 样报告基因是检测不同基因型中蛋白质丰度的敏感工具。然而，我们发现不同基因型中空的 StrigoQuant 载体的相对 FF/REN 比值存在显著差异。因此，为了确定实际的蛋白质丰度，应该将感兴趣的蛋白质的相对 FF/REN 比值与空载体的比值进行归一化。