An intein-based biosensor to measure protein stability in vivo.

Autor: Son A; Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, Colorado, USA., Smetana JS; Department of Biological Sciences, Murray State University, Murray, Kentucky, USA., Horowitz S; Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, Colorado, USA., Lennon CW; Department of Biological Sciences, Murray State University, Murray, Kentucky, USA.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2024 Mar; Vol. 33 (3), pp. e4925.
DOI: 10.1002/pro.4925
Abstrakt: Biosensors to measure protein stability in vivo are valuable tools for a variety of applications. Previous work has demonstrated that a tripartite design, whereby a protein of interest (POI) is inserted within a reporter, can link POI stability to reporter activity. Inteins are translated within other proteins and excised in a self-mediated protein splicing reaction. Here, we developed a novel folding biosensor where a POI is inserted within an intein, which is subsequently translated within an antibiotic resistance marker. We showed that protein splicing is required for antibiotic resistance and that housing a stable POI within the intein, compared to an unstable variant, results in a 100,000-fold difference in survival. Further, using a fluorescent protein that matures slowly as the POI, we developed a reporter with two simultaneous readouts for protein folding. Finally, we showed that co-expression of GroEL can significantly increase the activity of both reporters, further verifying that protein folding factors can act on the POI in the biosensor. As a whole, our work provides a new twist on the traditional tripartite approach to measuring protein stability in vivo.
(© 2024 The Protein Society.)
Databáze: MEDLINE
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