PENGARUH MUTASI DAERAH UPSTREAM DAN DOWNSTREAM KODON START PADA PLASMID PCAD2_RET DAN PMCD_RET TERHADAP EKSPRESI RETEPLASE

• Main Author: Wijaya, Christopher
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48617
• Institution: Institut Teknologi Bandung

Cardiovascular disease is a disorder in the functioning of the heart and blood vessels, such as heart attack (myocardial infarction) and ischemic stroke. This disease is the number one cause of death in the world. Treatment that can be applied is using thrombolytic agent, such as reteplase. At present, the production of reteplase has been carried out by a recombinant approach using prokaryotic cells (Escherichia coli). In E. coli gene expression system, a promoter is needed to initiate DNA transcription producing mRNA which is then converted into reteplase. In this research, the autoinduction promoters used were dps promoter (pCAD2_ret plasmid) and gadA (pMCD_ret plasmid). Site-Directed Mutagenesis (SDM) was performed on both plasmids expected to change the secondary structure of mRNA, so that the RBS and start codon regions is accessible, resulting in smooth translation initiation. SDM was also carried out in the upstream region of the pCAD2_ret plasmid start codon with deletion of GAT base from 15th to 17th base. Another SDM was carried out in the downstream region of the start codon by changing the pCAD2_ret and pMCD_ret bases on 5 th and 6th codon (CAC-5-CAT and CAT-6-CAC). SDM was carried out using Single-Primer Reactions in Parallel (SPRINP) method. The results of DNA sequencing showed that the mutation was only successful in the downstream region of the start codon for both plasmids. To see the effect of mutation, production of reteplase was carried out in E. coli TOP10 containing mutated pCAD2_ret plasmid on the CDS area under the following conditions: 37°C, 150 rpm, 24 hours, in 30 mL LB media. The result of the analysis of protein production using fast lysis with SDS-PAGE showed that the production of reteplase using E. coli TOP10 containing mutated pCAD2_ret plasmid on the CDS area has been successfully carried out in the presence of a protein band at a size of 40.9 kDa.