Design, Cloning, and Expression of PLC-Darpin Fusion Protein as Putative Immunotoxin in a Prokaryotic Host

Background & Objective:  Cancers are common genetic disease that can cause death. Bacteria, such as Clostridium novyi, potentially could be used in cancer treatment by producing an enzyme called phospholipase C (PLC), which causes cell lysis. The aims of this study are to cloning and expression of PLC- Darpin in prokaryotic host.  Materials & Methods:  Briefly, the PLC- Darpin gene sequence was amplified using PCR. The amplified fragment was conducted into the pET۲۸a vector, transformed into E. coli BL۲۱ (DE۳), and screened by the double digestion method. Protein expression was analyzed using SDS-PAGE and checked with specific antibodies using the western blotting method. The cloned fragment was confirmed using the PCR colony method. Results:  Designed PLC- Darpin protein sequence (۱۶۰۰ bp) was successfully amplified by specific primers taking advantage of PCR method. Then, cloned PLC- Darpin candidate sequence was reconfirmed by digestion procedure, and colony PCR. Finally, ۶۰ KDa expressed protein in prokaryotic host reconfirmed by SDS- page and western blotting. Conclusion:  Fused PLC enzyme to Darpin protein could be considered as a main putative immunotoxin due to its enzymatic role and cytotoxicity which may be utilized as a therapeutic choice in the future with further investigations.

Keywords:

Immunotoxin , Gene cloning , Darpin , Fusion protein , PLC-Darpin