Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen |
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Authors: | Hui Yao Liqun Wang Jia Guo Weimin Liu Jingjing Li Yingxiao Wang Linhong Deng Mingxing Ouyang |
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Institution: | 1 Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, 213164, China2 Department of Bioengineering, University of Illinois at Urbana-Champaign, Illinois, 61801, USA3 College of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou, 213164, China4 Changzhou City Second People’s Hospital, Changzhou, 213164, China |
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Abstract: | Prostate cancer is the most common cancer among men beyond 50 years
old, and ranked the second in mortality. The level of Prostate-specific 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 fluorescence resonance energy transfer (FRET) technology 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 reflecting the existence of other inhibitory factors besides Zn2+.
By expressing the biosensor on cell plasma membrane, the FRET responses were
significant, but couldn’t distinguish well the cultured prostate cancer cells from
non-prostate cancer cells under microscopy imaging, indicating insufficient speci-
ficity 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 inhibitors; the purified biosensor protein can also be an alternative choice for measuring PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate
from chemical synthesis. |
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Keywords: | Prostate-specific antigen fluorescence resonance energy transfer serine protease biosensor prostate cancer |
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