OPTIMATION EXPRESSION OF RECOMBINANT THERMOSTABLE ARCHAEAL DNA POLIMERASE B FROM METAGENOM

• Main Author: Yessy
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32760
• Institution: Institut Teknologi Bandung

DNA Polymerase (DNA Pol) is enzyme that catalyze DNA synthesize in organism. DNA Pol has been widely applied and has role in biotechnology especially genetic engineering research for example Polymerase Chain Reaction (PCR), sequencing and DNA labelling. One of steps in these processes is carried out at high temperature (90 - 95 °C) to denaturate double strand DNA to single strand DNA. In the beginning, these processes used thermolable DNA Polymerase but it will be denatured at high temperature so that it will be added at every cycles. Nowadays thermostable DNA is widely applied in these process than thermolable DNA Polymerase. In previous study, metagenom approach was carried out to isolate archaeal DNA Pol B from extreme environment Domas crater, Tangkuban Perahu, West Java (temperature 93 – 95 °C, pH 1-2). From that study 2,7 kilobase (kb) DNA pol B gene was obtained. From nucleotide sequence alignment, this gene has high homology (92%) with pol B Metallosphaera sedulla. This gene was cloned into expression vector pEt-30a(+) and theoretical molecular weight was determined (~101 kDa). But expression of this gene is not carried out yet, so that this research focuses to optimize expression2,7 kilobase DNA pol B gene. Parameters like temperature and isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration have roles in determining protein expression level. Optimation expression is carried out to determine in what condition protein expressed at high level. In this research, optimation expression is determined based on induction time and IPTG concentration. In this research induction time varied into 3,4,5 and 6 hour, and IPTG concentration varied into 0,0; 0,5; 1,0 dan 1,5 mM. Induction was carried when transforman cells reach OD600 ~0,6-0,8. Induction higher than OD 0,8 will gives low level expression because cells will stop growing immediately after too high density was reached. SDS-PAGE electroforegram showed 3-hour induction time gives higher expression level than other induction time. For 3-hour induction time, expression was higher if IPTG 1,5 mM was used, but for 4,5 and 6-hour induction time expression was higher if IPTG 0,5 mM was used. To determine level expression quantitatively, calculation with imageJ program was carried out. ImageJ program is program that can measure SDS-PAGE protein band intensity. Intensity showed peaks that represents each protein. From this calculations transforman cell showed higher level expression than transforman cell with no IPTG addition, 0,5 mM IPTG addition showeD expression level 10,67 %, compared with no IPTG addition (7,01 %). IPTG addition 1 mM and 1,5 mM showed expression level successively 11,64 % and 13,90 %. From all calculation, expression level of each protein showed highest level expression at 3-hour induction time and 1,5 mM IPTG concentration with level expression 13,90 %.