Construction Of A Novel Protease Biosensor Based On Fluorescence Resonance Transfer Technique

Background and Aim:Since the theory of fluorescence resonance energy transfer was established in 1948, FRET has be widely used in many fields. FRET is strongly dependent on the distance between the donor and acceptor. Therefore, FRET is widely used for the detection of intermolecular interactions. The discovery of Green fluorescent protein (GFP) from the jellyfish Aequorea Victoria and its many variants, which are quite suitable for the donor/acceptor pair of FRET have greatly accelerated the development of FRET. Recently, FRET- based measurement has become a novel detection method to monitor protein-ligand and protein-protein interactions. Since FRET-based biosensor has much higher specificity and sensitivity, it becomes one of the best systems for High Throughput Screen (HTS) and proteomic application.Prostate specific antigen (PSA) is normally secreted from prostate luminal epithelial cells with normal serum PSA level below 4.0ng/ml. Clinically, serum PSA level has been used as a sensitive marker to screen for prostate cancer. However, the serum PSA level may not be specific for prostate cancer as it may also be elevated in some patients with benign prostate diseases. Particularly when PSA level less than 10 ng/ml, it is hard to differentiate and diagnose. Some studies have shown that distinguishing and measuring different forms PSA has improved the specificity of diagnosis. One such variant, proteolytically active PSA, is a potentially useful tumor marker. But many of the currently available PSA detection methods suffer from the inability to distinguish the various forms of PSA. Therefore, it is of great importanceto develop some new methods to discriminate the different PSA isoforms, including the proteolytically active form.The aim of this study is to establish a fluorescence resonance energy transfer (FRET)-based biosensor for detecting the enzymatically active prostate-specific antigen (PSA) in vitro. And also to construct a cassette inserting prokaryotic expression vector of the substrate protein used in in vitro FRET measurement for the detection of protease activities. The protease Caspase-3 was selected for its evaluation.Methods:With the prokaryotic expression plasmid pET-CFP- SSYYSG -YFP which was constructed previously in this laboratory and can express the fusion protein in the E. coli strain BL21DE3 characterized with a PSA specific recognition sequence of SSYYSG between ECFP and EYFP, the purified fusion protein was obtained . The substrate protein was subjected for in vitro cleavage with chymotrypsin, trypsin, a purified enzymatically active form of PSA and seminal plasma. The changes of FRET signal were detected by fluorospectrophotometry.For construction of a cassette inserting prokaryotic expression vector of the substrate protein for FRET detection of protease activities, the PCR products of ecfp from the Clontech plasmid containing the genes of the ecfp were cloned into the pET30a(+)-YFP vector generating a new expression plasmid pET30a(+)-CFP-MCS-YFP into which we can insert the coding oligonucleotides of protease specific recognition peptide freely. To test it, we selected peptides DEVD which can be specifically recognized and cleaved by Caspase-3. The subsequent expression and purification of the fusion protein, but also in vitro proteolytic reaction and spectroscopic analysis was conducted basically the same as described above.Results:1. Chymotrypsin, as well as the purified enzymatically active form of PSA, was able to decrease the fluorescence emission of fusion protein ECFP-SSYYSG-EYFP at 527 nm in a dose- and time-dependent manner, while trypsin gave no FRETinhibition within 90 min. Furthermore, incubation of seminal plasma with the fusion protein ECFP-SSYYSG-EYFP could also inhibit FRET phenomena. 2. Sequence data showed that the encoding sequence of DEVD was successfully inserted between ECFP and EYFP coding sequences; the fusion protein ECFP-DEVD-EYFP can be cleaved by cell lysates prepared from the FL cells pretreated with MNNG at a apoptosis inducing concentration as shown by the...