A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time

Bahun, Miha (avtor), Poklar Ulrih, Nataša (avtor)

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

• Jezik: Angleški jezik
• Ključne besede: protease assay, proteolysis, subtilisin, SYPRO orange, fluorescence, real-time PCR, thermostability
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: BF - Biotehniška fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2025
• Št. strani: 9 str.
• Številčenje: Vol. 696, art. 115688
• PID: 20.500.12556/RUL-164279
• UDK: 577.152.34
• ISSN pri članku: 0003-2697
• DOI: 10.1016/j.ab.2024.115688
• COBISS.SI-ID: 212076803
• Datum objave v RUL: 18.10.2024

Gradivo je del revije

• Naslov: Analytical biochemistry
• Skrajšan naslov: Anal. biochem.
• Založnik: Elsevier
• ISSN: 0003-2697
• COBISS.SI-ID: 6506759

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: encimi, proteaze, aktivnost proteaz, proteoliza, PCR

Projekti

• Financer: ARIS - Javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije
• Številka projekta: P4-0121
• Naslov: Biokemijska in biofizikalno-kemijska karakterizacija naravnih snovi