SUBCLONED OF GENES ENCODING AMORPHA-4,11-DIENE SYNTHASE (ADS) AND CYTHOCHROME P450 MONOOXYGENASE (CYP71AVI) INTO pETDUET1 VECTOR FOR EXPRESSION IN Escherichia coli
Background and objectives: Artemisinin is an anti-malarial drug produced by Artemisia annua L. in small amount (between 0.001 - 1% of the dry weight). One of potential method to obtain artemisinin in large amount is addition of artemisinic acid (precursor artemisinin) biosynthesis pathway in Esch...
Saved in:
| Main Author: | |
|---|---|
| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/45760 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Background and objectives: Artemisinin is an anti-malarial drug produced by
Artemisia annua L. in small amount (between 0.001 - 1% of the dry weight). One
of potential method to obtain artemisinin in large amount is addition of
artemisinic acid (precursor artemisinin) biosynthesis pathway in Escherichia coli.
This can be done by genetic engineering to the enzymes involved in the
biosynthesis of artemisinic acid, such as amorpha-4,11-diene synthase (ADS) and
cytochrome p450 monooxygenase (CYP71AVI). In this study, gene encoding
ADS and CYP71AVI were subcloned into pETDUET1 vector in E. coli and then
the expression products characterized with SDS-PAGE and Western Blot.
Methods: Gen construction started with subcloning of CYP71AVI gene from
pJexpress401_cyp into pETDUET1 through restriction site NdeI and XhoI to get
pETDUET1_cyp. Subsequently, ADS gene isolated from pET15b_ads by PCR
and produced ADS gene fragment with restriction site of BamHI in 5’end and
NotI in 3’end. This fragment was cloned into intermediate vector pGEM-T to get
pGEM-T_ads. Then ADS gene from pGEM-T_ads was subcloned into
pETDUET1_cyp to get pETDUET1_cyp/ads. Confirmation of the recombinant
vector was done by migration, restriction site, and sequencing analysis.
Confirmed pETDUET1_cyp/ads transformed to E. coli BL21(DE3) for production
of CYP71AVI and ADS by induction of 0.5 mM IPTG at 28 °C for 6 hours and at
37 °C for 3 hours. The proteins were characterized with SDS-PAGE and Western
Blot. Results: pETDUET1_cyp, pGEM-T_ads, and pETDUET1_ cyp/ads were
constructed and confirmed by migration, restriction site, and sequencing analysis.
Both genes carried sequence of gene that encoding his-tag. CYP71AVI and ADS
protein have been produced in E. coli as inclusion body. The proteins were
identified as 55.78 kDa (CYP71AVI) and 64.04 kDa (ADS) protein in size. The
protein that was recognized by anti his-tag antibody in Western Blot analysis was
the 58.07 kDa protein (CYP71AVI) only.
|
|---|