STUDI LITERATUR SOLUBILISASI DAN PELIPATAN ULANG BADAN INKLUSI RETEPLASE REKOMBINAN HASIL EKSPRESI PADA ESCHERICHIA COLI

• Main Author: Exaudi Sitorus, Arjuna
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/56688
• Institution: Institut Teknologi Bandung

Cardiovascular disease is a disorder in heart and blood vessels function. Cardiovascular disease is also the number one cause of death in the world including in Indonesia. The highest prevalence is heart attack (myocardial infarction) and ischemic stroke. One of the treatment approved by FDA for these diseases is thrombolytic agents such as reteplase. However, reteplase is not available and has never been produced in Indonesia yet. Reteplase is a multidomain protein with nine disulfide bonds. Currently, the production of recombinant reteplase protein has been carried out in prokaryotic cells (Escherichia coli). Reteplase production in E. coli cells often forms insoluble aggregates that are pharmacologically inactive, also known as inclusion bodies. Various in vitro and in vivo methods and strategies are being developed to produce reteplase in both active and dissolved forms. In vitro strategies tend to be simpler. In vitro strategies generally consist of isolation, solubilization, refolding, and purification. Solubilization and refolding is the most responsible processes to the low yields of dissolved and active reteplase issues. In this literature study, various previous studies regarding in vitro strategies to produce reteplase in the dissolved and active form of reteplase inclusion bodies are reviewed. This literature study proposed methods of solubilization and refolding of reteplase inclusion bodies by associating the characteristics of reteplase inclusion bodies compared with other inclusion bodies that had been successfully solubilized or refolded and gave high yields. The proposed solubilization includes two mild solubilisation methods, namely a combination of medium base (pH ? 12), DTT, and low concentration of chaotrope such as urea and detergent-based solubilization such as sarkosyl. The proposed refolding method is to use urease-mediated refolding. Urease was previously immobilized on NHS-Sepharose resin so as not to add contaminants. The procedure followed by filtration and dialysis to remove resin and ammonia. Dialysis is carried out with the addition of tranexamic acid (Txa) and glycerol to the phosphate buffer solution. Dialysis was carried out once again to remove Txa and glycerol to obtain pure, active, and dissolved form of reteplase.