EVALUASI PRODUKSI RETEPLASE REKOMBINAN HASIL KOEKSPRESI DENGAN CHAPERONE PROTEIN DISULFIDE ISOMERASE PADA Escherichia coli BL21(DE3)

• Main Author: Auliya Mulyawan, Ditta
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40357
• Institution: Institut Teknologi Bandung

Reteplase is a recombinant therapeutic protein that belongs to recombinant plasminogen activator group (r-PA) and functions as a thrombolytic agent to treat myocardial infarction. Most of reteplase production carried out in Escherichia coli tends to form inclusion bodies, insoluble form of protein, since it has nine pairs of disulfide bonds. One attempt to improve solubility of reteplase is by using Protein Disulfide Isomerase (PDI) Chaperone. PDI catalyzes the formation of disulfide bonds in protein. The purpose of this study is to determine the effect of coexpression of recombinant reteplase with PDI on the production and solubility of recombinant reteplase, and determine its effect on the host cell’s growth profile. The host cell used in this study is E. coli BL21(DE3) carrying pET24b_ret and pT7-7_PDI. Plasmid carried by recombined E. coli was confirmed by using migration analysis and restriction analysis. The E. coli BL21(DE3) growth profile carrying pET24b_ret and pT7-7_PDI was compared with those that carrying each plasmid and those without insertion plasmid. Optimization of recombinant reteplase overproduction condition was carried out by modification of temperature (20 ?C and 37 ?C). Cell culture was induced when OD600 reached 0.6 –0.7 by isopropylthio- -D-galactosidase (IPTG) with a final concentration of 0.1 and 0.5 mM. Overproduced recombinant reteplase and PDI was characterized using SDS-PAGE. E. coli BL21(DE3) carrying pET24b_ret and pT7-7_PDI has a similar growth profile with the other E. coli BL21(DE3). Recombinant reteplase that coexpressed with PDI was produced more at 20 ?C with IPTG final concentration is 0.5 mM, but the reteplase was produced as inclusion body. In conclusion, coexpression recombinant reteplase with PDI did not increase the solubility of reteplase, and does not affect the host cell’s growth profile.