PENGARUH PROMOTOR dps TERHADAP PRODUKSI PROTEIN TROMBOLITIK PADA Escherichia coli

• Main Author: Atika, Friniva
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40440
• Institution: Institut Teknologi Bandung

Cardiovascular diseases, especially heart attack and stroke, are the major cause of death in the world. One effective treatment strategy to treat those diseases is by using thrombolytic agents in the form of therapeutic proteins such as plasminogen activators or plasmin-like proteins. Nowadays, there are a lot of thrombolytic agents produced by recombinant DNA technique. In this technique, promoter is a part of expression cassette in recombinant plasmid that plays an important role in the transcription process and can subsequently affect the amount and the form of expressed protein. The dps promoter is a type of autoinducible promoter that works under metabolic control of host cells. The use of autoinducible promoter gives some benefits especially in terms of culture handling and the cost of protein production. Based on promoter strength, dps promoter is categorized as weak promoter. Weak promoter produces protein slowly, hence the folding of protein will be more properly. This makes higher amount of soluble form of protein could be generated compared to production using strong promoter. The aim of this research is to determine the effect of dps promoter on trombolytic protein production by constructing pCAD2_DFEG169A plasmid and to evaluate the production of reteplase and Douchi Fibrinolytic Enzyme (DFEG169A) proteins using pCAD2_ret and pCAD2_DFEG169A plasmids containing dps promoters in Escherichia coli. In this research, construction of pCAD2_DFEG169A plasmid was successfully carried out, confirmed by plasmid migration and restriction analysis with agarose gel electrophoresis. Production of reteplase and DFEG169A were performed by culturing E. coli that carrying each plasmid in LB media at 37?C for 24 hours. The result of protein overproduction which is analyzed by SDS- PAGE did not showed thick protein band at the size of reteplase (39.6 kDa) and DFEG169A (28 kDa). In conclusion, dps promoter cannot be used to produce reteplase in E. coli ???????? ?¸ E. coli Rosettagami, as well as DFEG169A in E. coli TOP10 at expression system and condition used in this research.