Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen

: Prostate cancer is the most common cancer among men beyond 50 years old, and ranked the second in mortality. The level of Prostate-speci fi c antigen (PSA) in serum has been a routine biomarker for clinical assessment of the cancer development, which is detected mostly by antibody-based immunoassays. The proteolytic activity of PSA also has important functions. Here a genetically encoded biosensor based on fl uorescence resonance energy transfer (FRET) tech-nology was developed to measure PSA activity. In vitro assay showed that the biosensor containing a substrate peptide ‘ RLSSYYSGAG ’ had 400% FRET change in response to 1 µg/ml PSA within 90 min, and could detect PSA activity at 25 ng/ml. PSA didn ’ t show enzymatic activity toward the biosensor in serum solution, likely re fl ecting the existence of other inhibitory factors besides Zn 2+ . By expressing the biosensor on cell plasma membrane, the FRET responses were signi fi cant, but couldn ’ t distinguish well the cultured prostate cancer cells from non-prostate cancer cells under microscopy imaging, indicating insuf fi cient speci- fi city to PSA. The biosensor with the previously known ‘ HSSKLQ ’ substrate showed little response to PSA in solution. In summary, we developed a genetically encoded FRET biosensor to detect PSA activity, which may serve as a useful tool for relevant applications, such as screening PSA activation substrates or inhi-bitors; the puri fi ed biosensor protein can also be an alternative choice for measur-ing PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate from chemical synthesis.