title: Use of hGluc/tdTomato pair for sensitive BRET sensing of protease with high solution media tolerance.
abstract: Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Fengyun
authors.initials: F
authors.lastname: Li
authors.forename: Junping
authors.initials: J
authors.lastname: Yu
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zongqiang
authors.initials: Z
authors.lastname: Cui
authors.forename: Dianbing
authors.initials: D
authors.lastname: Wang
authors.forename: Hongping
authors.initials: H
authors.lastname: Wei
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
mesh_terms: D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000295:instrumentation / Q000379:methods*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004765:Enteropeptidase / Q000097:blood; D057230:Limit of Detection; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012996:Solutions; D000097573:Red Fluorescent Protein
chemical_list: D004765:Enteropeptidase; D008156:Luciferases; D008164:Luminescent Proteins; D010447:Peptide Hydrolases; D011993:Recombinant Fusion Proteins; D012996:Solutions; D000097573:Red Fluorescent Protein