Design and Development of Luciferase Based Biosensor to Detect Caspase-9 Activity

Caspase family are involved in inflammation, apoptosis and cell differentiation. Caspase-9, is a key enzyme in apoptosis and recently evidences has suggested that Caspase-9 activity has a pivotal role in determining cell fate. Therefore, monitoring Caspase-9 activity would be good indicator of stem cell differentiation and apoptosis. At present study, we have designed and developed a bioluminescent sensor to detect stem cell differentiation and apoptosis based on caspase-9 activity. It is composed of a caspase-9 ligand flanked by two N and C domain of Renilla luciferase, in reverse orientation. Following the biosensor cleavage, luciferase folds into its correct shape and its bioluminescent activity is triggered which can be detected. The first step of designing and construction of Circular Permutated Renilla luciferase (CPR) was in silico structural studies of new designed CPR and its comparison with its primary form. This step was performed by I-TASSER serve. Engineered CPR gene was produced using SOE PCR. CPR gene was cloned into expression vector. Recombinant vector was transformed in to a replicative host and cultured. Positive colonies were selected using colony-PCR. The recombinant vector was extracted. Digestion of recombinant vector were performed and finally cloning was confirmed by sequencing analysis. Recombinant plasmid retransformed into an expression host. SDS-PAGE gel electrophoresis of expressed recombinant protein under different conditions showed the best condition of expression. Recombinant protein purified using affinity chromatography. Finally, CPR activity measurement at the presence and absence of Caspase-9 showed that CPR produces higher intensity of light during caspase-9 activity.