The Development of a High Sensitive FRET-based Protease Assay
for Kinetic Determination of SUMO-specific Protease – SENP1
Yan Liu1, Yang Song1, Vipul Madahar1, Jiayu Liao1, 2,*
Department of Bioengineering and 2Institute for Integrative Genome Biology, Bourns College of Engineering,
University of California at Riverside, 900 University Avenue, Riverside, CA 92521
Tel: 951-827-6240 Email:jliao@engr.ucr.edu
1
Summary: A high sensitive FRET-based protease assay has been developed to study
kinetic properties of SENP1’s SUMO1 maturation. Quantitative analysis of FRET signal has been
set up. kcat/KM = (2.9856  0.5669)  107 (M-1s-1) was calculated by using this protease assay and
developed methodology, which is consistent to previous studies.
Förster Resonance Energy Transfer (FRET) technology has been widely used in biological and biomedical research
and is a very powerful tool in elucidating protein interactions in many cellular processes. SUMOylation is an
important post-translational protein modification mechanism which plays an important role in a variety of biological
processes. Conjugation of SUMO is a cascade event requiring multiple protein-protein interactions [1]. SUMOspecific protease (SENP) either functions as an endopeptide to process the pre-SUMO or as an isopeptide to
deconjugate SUMO from its substrate [2].
Engineered fluorescent protein CyPet (donor) and YPet (acceptor) were genetically tagged on N- and C- terminus of
pre-SUMO1 respectively. The recombinant fusion protein CyPet-(pre-SUMO1)-YPet was mixed with catalytic
domain of SENP1. The SUMO1’s tail was removed with YPet by SENP1. The maturation process was monitored by
the disrupted energy transfer from CyPet to YPet. The maximum reaction velocity (Vmax) and KM were obtained from
Mechalies-Menten equation.
To elucidate the advantage of CyPet-YPet FRET pair, we compared the ratiometric change of emission at YPet’s
peak (530nm) to CyPet’s peak (475nm) to that of CFP-YFP. The result showed that CyPet-YPet as FRET pair
provides higher energy transfer efficiency and more sensitive to the protease digestion, the ratio of fluorescent
changed from 4.23 to 0.63 treated by SENP1, while the ratio of CFP-YFP pair changed from 1.1 to 0.49. As 7 SENP
family members and 4 SUMO paralogs have been found in human genome. The developed protease assay can be
applied to study different specificities of SENP paralog to various SUMO members. The fluorescent emission at
530nm decreased 85% when treated with SENP1, and only decreased 1% when treated with SENP7, which suggests
high substrate specificity. Kinetic study is a common way to quantitatively characterize the catalytic specificities of
different substrate-protease pair. Fluorescent emission at 530nm were divided into three fractions: donor’s direct
emission, acceptor’s director emission and FRET signal, each fluorescent contribution were quantitatively related to
the protein concentration; enzyme kinetics study presented that KM=0.735 uM and kcat=21.944  4.1666 (s-1), kcat/KM
was (2.9856  0.5669)  107 (M-1s-1), which is consistent to previous studies [3].
The new developments include theoretical and experimental procedures in determination of catalytic efficiency
(kcat/KM) of catalytic domain SENP1 towards SUMO1. The general principle of this high sensitive in vitro FRETbased protease assay could be applicable to study and compare specificities of various SENPs towards different preSUMO paralogs as well as other proteases.
[1] Johnson, E.S., Protein modification By SUMO. Annual Review of Biochemistry, 2004. 73(1): p. 355-382.
[2] Drag, M. and G.S. Salvesen, DeSUMOylating enzymes-SENPs. IUBMB Life, 2008. 60(11): p. 734-742.
[3] Shen, L., et al., SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nature Structural & Molecular Biology, 2006.
13(12): p. 1069-1077.