EVALUASI PRODUKSI RETEPLASE REKOMBINAN HASIL KOEKSPRESI DENGAN CHAPERONE PROTEIN DISULFIDE ISOMERASE PADA Escherichia coli BL21(DE3)
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 protei...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/40357 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | 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.
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