OPTIMIZATION OF SOLUBILIZATION INCLUSION BODIES AND PURIFICATION OF RECOMBINANT AMORPHA-4,11-DIENE SYNTHASE
Artemisinin is an antimalarial drug that were used as combination therapy to overcome chloroquine resistancy. Artemisinin was found in Artemisia annua at low level, about 0.01-0.5%. One of biotechnological approach to increasing artemisinin levels is by producing the enzymes that were involved in...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/45371 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Artemisinin is an antimalarial drug that were used as combination therapy to
overcome chloroquine resistancy. Artemisinin was found in Artemisia annua at
low level, about 0.01-0.5%. One of biotechnological approach to increasing
artemisinin levels is by producing the enzymes that were involved in artemisisnin
biosynthesis in Eschericia coli. Enzyme that has important role in artemisinin
biosynthesis is amorpha-4,11-diene synthase (ADS), has been produced in E. coli
BL21(DE3) as recombinant protein (rADS) but dominant as inclusion body (IB)
which is insoluble and inactive. To activate the rADS-IB, solubilization and
refolding process are needed. The aim of this study are to optimize the
solubilization of rADS-IB and to purify rADS. Solubilization of inclusion body
was done by pH modification and denaturant addition (urea and guanidine-HCl).
Purification and refolding of rADS-IB were done in column using Ni-NTA resin.
Purification of soluble rADS was performed by single step purification (affinity
chromatography) and multistep purification (gel filtration and ion exchange
chromatography). Solubilization IB with pH modification showed that only 2.3%
(w/w) rADS-IB were solubilized from total IB. Solubilization with denaturant
showed that rADS-IB does not soluble in various concentrations of urea (2, 4, 6,
8M) but completely soluble in 6M guanidine-HCl. On column refolding and
purificatiaon of solubilised rADS-IB by affinity chromatography with 6M urea as
washing buffer had caused rADS aggregation in column. The best result was
achieved by 6M guanidin-HCl and Triton X-100 as washing buffer with 90%
rADS purity level and 65.07% yield. Purification of soluble rADS shown that
rADS did not bind well to the Ni-NTA and Ni-TED resin in contrast to multistep
purification which succesfully purify rADS at purity level 94.7% and the yield is
58%.
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