Overcoming fluorescence loss in mEOS-based AAA+ unfoldase reporters through covalent linkage

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-04-28 DOI:10.1016/j.pep.2025.106724

Isabella R. Walter , Baylee A. Smith , Dominic Castanzo , Matthew L. Wohlever

引用次数: 0

Abstract

Recent work has demonstrated that the soluble photoconvertable fluorescent protein mEOS can be a reporter for AAA+ (ATPases Associated with diverse cellular Activities) unfoldase activity. Given that many AAA+ proteins process membrane proteins, we sought to adapt mEOS for use with membrane protein substrates. However, direct genetic fusion of mEOS to a membrane protein completely abolished fluorescence, severely limiting the utility of mEOS for studying AAA+ proteins. To circumvent this challenge, we separately purified mEOS and multiple different AAA+ degrons, including a transmembrane domain. We then covalently linked mEOS and the degrons via Sortase. This innovative approach preserves mEOS fluorescence and photoconversion, even upon linkage to a transmembrane domain. Together, this work offers a broadly applicable platform for the study of membrane associated AAA+ proteins.

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通过共价连锁克服基于meos的AAA+展开酶报告基因的荧光损失

最近的研究表明，可溶性光转化荧光蛋白mEOS可以作为AAA+（与多种细胞活性相关的atp酶）展开酶活性的报告蛋白。考虑到许多AAA+蛋白加工膜蛋白，我们寻求将mEOS用于膜蛋白底物。然而，mEOS与膜蛋白的直接遗传融合完全消除了荧光，严重限制了mEOS在研究AAA+蛋白中的应用。为了克服这一挑战，我们分别纯化了mEOS和多个不同的AAA+ degrons，包括一个跨膜结构域。然后我们通过排序酶将mEOS和degrons共价连接。这种创新的方法保留了mEOS的荧光和光转化，甚至连到跨膜结构域。总之，这项工作为研究膜相关的AAA+蛋白提供了一个广泛适用的平台。

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and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.