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

Hui Yao1, Liqun Wang3, Jia Guo1, Weimin Liu4, Jingjing Li1, Yingxiao Wang2, Linhong Deng1,*, Mingxing Ouyang1,2,3,*
1 Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, 213164, China
2 Department of Bioengineering, University of Illinois at Urbana-Champaign, Illinois, 61801, USA
3 College of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou, 213164, China
4 Changzhou City Second People’s Hospital, Changzhou, 213164, China
* Corresponding Authors: Linhong Deng. Email: ; Mingxing Ouyang. Email:

Molecular & Cellular Biomechanics 2020, 17(3), 101-111. https://doi.org/10.32604/mcb.2020.09595

Received 05 January 2020; Accepted 22 March 2020; Issue published 01 July 2020

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.

Keywords

Prostate-specific antigen; fluorescence resonance energy transfer; serine protease; biosensor; prostate cancer

Cite This Article

Yao, H., Wang, L., Guo, J., Liu, W., Li, J. et al. (2020). Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen. Molecular & Cellular Biomechanics, 17(3), 101–111.

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This work is licensed under a Creative Commons Attribution 4.0 International License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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