Life Sciences & Biotechnology
Title : | Development of fluorescent human transmembrane serine protease 2 (TMPRSS2) activity sensor for rapid screening of inhibitors in vitro and in living cells to combat human COVID-19 and other respiratory infections. |
Area of research : | COVID-19 Research, Life Sciences & Biotechnology |
Focus area : | COVID-19 Diagnostics |
Principal Investigator : | Dr Vinay Bulusu, Indian Institute of Science Education and Research (IISER) Berhampur |
Timeline Start Year : | 2020 |
Contact info : | vbulusu@iiserbpr.ac.in |
Details
Executive Summary : | This study aims to develop a genetically encoded FRET sensor for TMPRSS2 containing a CFP-substrate-YFP module that can be tested under in-vitro and also in living cells.
The project proposes to do following experiments:
1. Generation of mTurquoise-hemagglutinin-Venus backbone from mTurquoise-Pdhr-Venus backbone reported previously by sub-cloning. Hemagglutinin (HA) has already been obtained from Prof Raghavan Varadarajan’s Laboratory, IISc, Bengaluru, and will be cloned soon.
2. Catalytic domain of hTMPRSS2 will be expressed and purified. Human TMPRSS2 plasmid is available in Addgene (Addgene # 53887) and will be obtained for subcloning.
3. hTMPRSS2 will be assayed using the mTurquoise-HA-Venus substrate and cleavage monitored by fluorescence spectroscopy. Validation will be done by TMPRSS2 inhibitors.
4. The FRET backbone will be expressed in HEK 293 cells and TMPRSS2 activity will be monitored that will be further validated by over expressing and knocking out TMPRSS2. |
Outcome/Output: | TMPRSS2 target protein H1N1 hemagglutin in full length and peptide sequence flanking the cleavage site are cloned in a FRET plasmid backbone. hTMPRSS2 ORF and its serine protease domain have been cloned in a bacterial expression plasmid. Expression analysis post IPTG induction showed a good expression of serine protease domain. A cell culture model to study SARS-COV-2 infection is being established using pseudovirions. |
Organizations involved