title: Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations.
abstract: A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.
authors.affiliation: University Hospital Centre of Vaud (CHUV), Epalinges, Switzerland.
authors.forename: Loyse M
authors.initials: LM
authors.lastname: Felber
authors.forename: Sylvain M
authors.initials: SM
authors.lastname: Cloutier
authors.forename: Christoph
authors.initials: C
authors.lastname: Kündig
authors.forename: Tadaaki
authors.initials: T
authors.lastname: Kishi
authors.forename: Vincent
authors.initials: V
authors.lastname: Brossard
authors.forename: Patrice
authors.initials: P
authors.lastname: Jichlinski
authors.forename: Hans-Jürg
authors.initials: HJ
authors.lastname: Leisinger
authors.forename: David
authors.initials: D
authors.lastname: Deperthes
mesh_terms: D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry
chemical_list: C485182:Cyan Fluorescent Protein; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases