PRODUCTION, PURIFICATION AND ACTIVITY TEST OF THERMUS AQUATICUS DNA POLYMERASE IN ESCHERICHIA COLI BL21-CODONPLUS(DE3)-RIPL

• Main Author: Puspawati, Fitria
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/56669
• Institution: Institut Teknologi Bandung

DNA polymerase (DNA pol) is enzyme that catalyzes DNA synthesis. DNA pol has been widely applied and has a role in biotechnology for Polymerase Chain Reaction (PCR) and quantitative PCR (qPCR). DNA polymerase Thermus aquaticus (DNA pol Taq) used in PCR has polymerase and exonuclease activity 5’?3'.The use of DNA pol Taq in Indonesia is quite high, but as an imported product. The aim of this research is to determine the optimal conditions for overproduction and purification DNA pol Taq, as well as to examine its polymerase activity. The synthetic DNA sequences of DNA Pol Taq has characteristics of resistance to PCR inhibitors with amino acid substitution at E626K, I707L, and E708N. In this study, open reading frame (ORF) encoding DNA pol Taq was synthesized and inserted into expresion vector pET28a. pET28a_DNA pol Taq was transformed into Escherichia coli BL21-CodonPlus(DE3)-RIPL. Overproduction results showed optimal conditions in IPTG induction with a final concentration of 0.1 mM, 18 hours at 37°C with soluble protein 64.23%. Pure protein is found more in protein produced at 37°C with imidazole concentration in the elution solution of 250-500 mM using Ni-NTA resin. DNA pol Taq was able to amplify PCR product until 3708 bp and showed that the enzyme can be used in molecular detection by qPCR and RT-qPCR. Specific activity of DNA pol Taq was 25000 U/mg. Performance of DNA pol Taq on PCR inhibitor showed that the DNA pol Taq is resistant against 0.1% SDS, 100 mM guanidine HCl, 8% blood plasma, 4% whole blood and 30%VTM. In conclusions, DNA pol Taq can be produced in the active form using pET28a_DNA pol Taq expression in E. coli BL21-CodonPlus(DE3)- RIPL and is resistant against tested inhibitor.