OVERPRODUCTION, PURIFICATION, AND POLIMERIZATION ACTIVITY TEST RECOMBINANT DNA POLYMERASE THERMUS THERMOPHILUS

• Main Author: Pratiwi Gunawan, Annisa
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/54339
• Institution: Institut Teknologi Bandung

DNA polymerase enzyme is one of the main components in polymerase chain reaction (PCR) because of its main role in synthesizing new DNA strands. This enzyme can be used in PCR and quantitative PCR (qPCR). One of the DNA polymerase enzymes used in PCR is DNA polymerase from Thermus thermophilus (DNA pol Tth). DNA pol Tth has exonuclease activity 5'?3' and does not have exonuclease activity 3 '?5' (low proof reading capacity). However, the use of DNA pol of this type is quite high and in Indonesia it is still imported. The aim of this research is to determine the optimal conditions for overproduction and purification DNA pol Tth which is produced recombinantly in Escherichia coli, as well as to carry out its activity tests. Overproduction of recombinant DNA pol Tth in E. coli BL21-CodonPlus (DE3)-RIPL (E. coli BL21 (DE3)-RIPL) was optimized with the use of media (Luria-Bertani (LB) and Terrific broth (TB)), temperature (25 and 37 °C), and induction time (3 and 20 hours). Overproduced proteins were characterized using SDS-PAGE analysis. Purification was carried out with Ni- NTA resin and the imidazole concentration was optimized in the elution solution. The activity test was performed using the PCR and qPCR methods. Overproduction results showed optimal conditions in LB medium, IPTG induction with a final concentration of 0.1 mM for 20 hours at 25 °C. Pure protein is found more in protein produced at 25° C with a concentration of imidazole in the elution solution of 100-500 mM. Polymerization activity test on DNA pol Tth enzyme gave positive results and showed that the enzyme can be used in molecular detection by conventional PCR and qPCR method using DNA template.