CONSTRUCTION OF GEN ENCODING DOUCHI FIBRINOLYTIC ENZYME MUTANT AND NATTOKINASE INTO PCAD2 PLASMID AND ITS EXPRESSION IN ESCHERICHIA COLI

• Main Author: Jeli Ping, Tirza
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/53183
• Institution: Institut Teknologi Bandung

The development of thrombolytic agents from food is becoming popular today because more effective and economies. Douchi Fibrinolytic Enzyme (DFE) and Nattokinase are examples of thrombolytic agents from fermented food microbes. Thrombolytic agents are needed for treating cardiovascular disease or ischemic stroke, which its incidence tends to increase in the world or Indonesia. This study is aimed to produce recombinant thrombolytic agent Douchi Fibrinolytic Enzyme mutant (DFEG169A) and Nattokinase in Escherichia coli using the alternative autoinducible promoter dps. pCAD2R_DFEG169A and pCAD2_NatWT plasmids carrying the dps promoter were constructed using quick step PCR and ligation techniques. Confirmation of correct construction was performed by migration, restriction analysis, and sequencing. The protein DFEG169A and Nattokinase were produced in E. coli Top10 and DH5? as a host cell, which is carrying the plasmids pCAD2R_DFEG169A and pCAD2_NatWT at an incubation time of 24 hours and 48 hours. Characterization of protein production was done by SDS-PAGE. Activity assay of the protein was carried out by radial caseinolysis method. In this research, construction of pCAD2R_DFEG169A and pCAD2_NatWT plasmids is successfully done. The plasmids size are 4812 bp and 4819 bp, respectively and positively confirmed by migration and restriction analysis using XbaI, XhoI, and BamHI. The DFEG169A and Nattokinase proteins were successfully produced recombinantly using that expression system in E. coli Top10 and DH5?in the form of preprotein (41 kDa) and mature protein (28 kDa) at 24 and 48 hours incubation time. Preliminary activity assay using radial caseinolysis method need to be confirmed further. In conclusion, autoinducible dps promoter can be used to produce Nattokinase protein recombinantly in E. coli.