OPTIMASI EKSPRESI ENZIM REVERSE TRANSCRIPTASE DARI MOLONEY MURINE LEUKEMIA VIRUS YANG DIFUSIKAN DENGAN PROTEIN SIS7A DARI SULFOLOBUS ISLANDICUS PADA SUHU INKUBASI 37?

• Main Author: Alita Fananda, Amalia
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/66156
• Institution: Institut Teknologi Bandung

Reverse transcriptase (RT) is a type of polymerase enzyme that has the ability to synthesize DNA with an RNA template. One of the commonly used RT enzymes is the RT enzyme from Moloney Murine Leukemia Virus (MMLV RT). It is known that MMLV RT has a higher catalytic activity and fidelity than other RT enzymes. However, some of its characteristics still require improvement, for example, its thermostability and processivity. In a previous in silico study, MMLV RT was fused with one of the members of the 7 kDa DNA-binding protein family from Archaea, namely Sis7a. This protein is thermostable and capable to bind to DNA. The combination of the two proteins is expected to improve MMLV RT’s performance, especially its processivity and its ability to maintain activity at higher temperatures. However, to validate the result of in silico analysis, this recombinant enzyme must be expressed in vitro. This study aims to find optimal conditions in expressing recombinant enzymes by determining the parameters of expression condition, which are inducer concentration and duration of induction. The constructed plasmid was cloned into Escherichia coli DH5?. Then, the transformation of cloned plasmid into E. coli BL21 (DE3) was conducted. The success of the transformation was confirmed through the method of PCR and DNA sequencing. The success of the transformation was confirmed by PCR and DNA sequencing. Protein expression was carried out at the optimum growing temperature of recombinant bacteria (37oC), using the combination of duration parameters (3, 4, and 5 hours), with the concentration of the inducer IPTG (0 mM, 0.25 mM, 0.50 mM, and 0.75 mM). Recombinant bacterial cultures were harvested, followed by fractionation of soluble and unsoluble proteins. The result of the protein expression was analyzed using SDS-PAGE method. The protein bands obtained were processed using ImageJ software and analyzed statistically. The results of PCR confirmation and DNA sequencing showed that the transformation was successfully performed in E. coli DH5? and E. coli BL21 (DE3). In E. coli BL21 (DE3), recombinant proteins were expressed in both soluble and insoluble fractions. In the soluble fraction, variations in induction duration exerted a statistically significant influence (p<0.05), while variations in IPTG concentrations did not provide a significant difference (p>0.05). The best induction for proteins of the soluble fraction was conducted in the course of 3 and 4 hours. In the unsoluble fraction, both variations in duration and IPTG concentrations, both exert a significant influence to the amount of recombinant proteins produced. Thus, it can be concluded that in this study has been successfully expressed the enzyme MMLV RT fused with Sis7a. Induction conditions were selected at a temperature of 37oC with duration of 4 hours, without the use of IPTG to obtain enzymes in the soluble fraction. Further research should be carried out for characterization of the expressed recombinant enzymes.