title: Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease.
abstract: Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.
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; D060829:Arthropod Proteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D002021:Buffers; D002413:Cations, Divalent; D004765:Enteropeptidase / Q000097:blood*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D060829:Arthropod Proteins; D001426:Bacterial Proteins; D002021:Buffers; D002413:Cations, Divalent; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases; D004765:Enteropeptidase