KLONING DAN OVEREKSPRESI DNA PENGKODE SUBTILISIN DFE PADA Escherichia coli BL21(DE3)

• Main Author: Darojatin, Ilma
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45386
• Institution: Institut Teknologi Bandung

Background and objectives: Subtilisin DFE is one of the fibrinolytic enzymes that is potential as a thrombolytic agent. This enzyme has high specificity to degrade fibrin directly and is able to hydrolize thrombus in vitro without damaging red blood cells. Subtilisin DFE open reading frame (ORF) has been isolated from Indonesian traditional fermented food in Pharmaceutical Biotechnology Laboratory, Bandung Institute of Technology. This study aims to clone and to overexpress subtilisin DFE open reading frame in Escherichia coli BL21(DE3) as well as to confirm proteolytic activity of the expressed product. Methods: ORF of subtilisin DFE that had been inserted in pGEM-T easy vector was moved into pET-16b expression vector by ligation based cloning method. The ligation reaction was transformed into E. coli TOP10 which then called pET-16b- DFE. This inserted plasmid was confirmed by analysis of migration, restriction, PCR, and DNA sequencing. The confirmed plasmid was transformed into E. coli BL21(DE3) to be overexpressed at a 150 mL scale in non-induction and induction with 0.1 mM IPTG for 3 hours incubation at 37 °C. The cell extract was then analyzed to determine the existence of target protein and its proteolytic activity. Results: ORF of subtilisin DFE had been successfully inserted into pET-16b vector and confirmed. Subtilisin DFE produced in E. coli BL21(DE3) was found as a mature soluble protein with 27.9 kDa molecular weight and showed caseinolytic activity. Conclusion: Cloning and overexpression of open reading frame of subtilisin DFE into E. coli BL21(DE3) had been successfully done and its expressed product had proteolytic activity.