#TITLE_ALTERNATIVE#

• Main Author: MAHARDIKA FORENTIN NIM: 20716011, ALFIAN
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/25339
• Institution: Institut Teknologi Bandung

Reteplase recombinant is a recombinant plasminogen activator (rPA) of alteplase<br /> derivatives used as thrombolytic for the treatment of acute myocardial infarction.<br /> Reteplase has been approved by FDA and EMA since 1996. However, reteplase has<br /> never been produced or marketed in Indonesia until now and has an expensive price.<br /> Reteplase is produced in Escherichia coli and tends to form inclusion body because<br /> it has nine disulfide bonds. The objective of this study was to optimize the dissolved<br /> reteplase production process using pET24b expression vector in E. coli<br /> BL21(DE3), to examine the activity and purify it with Ni-NTA affinity<br /> chromatography. Synthetic DNA coding codon optimized reteplase was brought by<br /> plasmid pET24b and referred as pET24b_ret. The production of reteplase was<br /> performed in recombinant E. coli BL21(DE3) carrying the plasmid with and<br /> without a pGroElDnaK chaperone plasmid. The recombinant E. coli was confirmed<br /> by the validity of it’s carried plasmid through the restriction analysis and<br /> sequencing of DNA encoding reteplase, GroEl and DnaK. Optimization of<br /> overproduction condition of reteplase protein was done by modify the temperature<br /> (20, 25, 37 and 40°C), Luria Bertani media composition (LB and ¼LB) and<br /> expression of reteplase in E. coli BL21(DE3) with or without pGroElDnaK. The<br /> culture was induced by isopropylthio-&#946;-D-galactoside with a final concentration of<br /> 0.05 mM at OD600 of 0.7-0.8. The overproduced reteplase was characterized using<br /> SDS-PAGE and analyzed with ImageJ software. Reteplase activity was determined<br /> with a tPA human chromogenic activity assay. The soluble form of produced<br /> reteplase was 1.14 ± 0.01 mg/g of wet cells and provided an activity of 45.97 IU/mg<br /> crude reteplase using production conditions at 20°C in recombinant E. coli without<br /> chaperones in LB media. While the highest insoluble of reteplase was produced at<br /> 37°C in recombinant E. coli without chaperones in LB media that resulting 19.6 ±<br /> 1.6 mg protein/g wet cells. The insoluble form of reteplase was successfully<br /> dissolved by 8 M urea, 0.1 M DTT in 50 mM pH 8 buffer Tris-Cl, and giving<br /> activity of 1.53 IU/mg crude reteplase but did not bind to Ni-NTA column. It can<br /> be concluded that reteplase can be produced in the active form using a plasmid<br /> pET24b_ret expression in E. coli BL21(DE3).<br /> <br />