SUBCLONED OF GENES ENCODING AMORPHA-4,11-DIENE SYNTHASE (ADS) AND CYTHOCHROME P450 MONOOXYGENASE (CYP71AVI) INTO pETDUET1 VECTOR FOR EXPRESSION IN Escherichia coli

• Main Author: Adi Wicaksono, Imam
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45760
• Institution: Institut Teknologi Bandung

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.