Design and Development of Biosensor for Detection of Cancer Stem Cells

Cancer Stem Cell (CSC) hypothesis suggests that a small subset of cancer cells have the ability to start and spread of tumors and are also able to metastasis and recurrence of the tumor. Extensive studies focusing on finding and introducing new CSC-specific markers to detect and study this subpopulation of cancer cells. 26s Proteasome enzyme activity which is involved in repairing DNA, cell cycle, cell growth and survival recently has been known as a CSC-marker. Here we report designing and developing of a Luciferase-based biosensor which works based on 26s Proteasome enzyme activity. Since 26s Proteasome activity in CSCs is lower than the other cancerous cells, it is expected that 26s Proteasome substrates are degraded in a large population of cancerous cells while they remain intact in CSCs. According to this idea, our strategy was conjugating a signal peptide, which was the substrate of 26s Proteasome, to the luciferase enzyme using protein engineering methods. Recombinant gene was constructed using PCR and then cloned into a vector. Recombinant vector was transformed into a replicative host and cultured. Positive colonies were selected using colony-PCR. The confirmed recombinant vector was transformed into an expression prokaryotic host. Upon different conditions of expression induction, recombinant protein expression level was evaluated using SDS-PAGE gel electrophoresis. The optimal conditions were set up, so the biosensor was expressed in high yield and it was purified using chromatography. Finally, the bioluminescent activity of biosensor was measured in vitro. The measurement results showed that the biosensor worked well and could be applicable in detection, isolation and monitoring CSCs.