A novel robust quantitative Förster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates.
SUMOylation (the process of adding the SUMO [small ubiquitin-like modifier] to substrates) is an important post-translational modification of critical proteins in multiple processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate the SUMO from its substrate. Determining the kinetics of SENPs is important for understanding their activities. Förster resonance energy transfer (FRET) technology has been widely used in biomedical research and is a powerful tool for elucidating protein interactions. In this paper we report a novel quantitative FRET-based protease assay for SENP2 endopeptidase activity that accounts for the self-fluorescent emissions of the donor (CyPet) and the acceptor (YPet). The kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel design. Although we use SENP2 to demonstrate our method, the general principles of this quantitative FRET-based protease kinetic determination can be readily applied to other proteases.
Department of Bioengineering, Bourns College of Engineering, University of California, Riverside, 900 University Avenue, Riverside, California 92521, USA. jiayu.liao@ucr.edu.
Yan
Y
Liu
Department of Radiation Oncology, Cancer Center, West China Hospital, Sichuan University, Chngdu 610041, P. R. China.
Yali
Y
Shen
Department of Health Sciences, College of Allied Health, California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504, USA.
Shasha
S
Zheng
Department of Bioengineering, Bourns College of Engineering, University of California, Riverside, 900 University Avenue, Riverside, California 92521, USA. jiayu.liao@ucr.edu and Institute for Integrative Genome Biology, University of California, Riverside, 900 University Avenue, Riverside, California 92521, USA.
Jiayu
J
Liao
D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D058207:Sumoylation
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases