BIOINFOMATIC STUDY OF PLASMODIUM FALCIPARUM LACTATE DEHYDROGENASE PROTEIN AND PLASMODIUM FALCIPARUM LACTATE DEHYDROGENASE CODING GENE EXPRESSION IN ESCHERICHIA COLI BL21(DE3)

• Main Author: Molani Alfinta Siahaan, Claudia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49347
• Institution: Institut Teknologi Bandung

Malaria is a contagious disease caused by the Plasmodium parasite. Malaria infection is transmitted through the bite of Anopheles sp. female. One of the problems related to malaria prevention is resistance to antimalarial drugs. Inappropriate use of antimalarial drugs due to inaccurate detection of parasites can lead to resistance. Rapid Diagnostic Test (RDT) or Rapid Diagnostic Test helps diagnose patients who are suspected of having malaria by detecting the presence of parasites in the blood. Plasmodium falciparum Lactate Dehydrogenase (PfLDH) is a protein that can be used as a biomarker in malaria RDT because it is produced in large numbers by parasites at the asexual and sexual stages. The malaria RDT circulating in the market is still imported. For this reason, local malaria RDT products need to be developed. As an initial stage in this research, a bioinformatics study was carried out on the PfLDH protein. The bioinformatics study includes prediction of secondary structures using SAS (Sequence Annotated by Structure) software. From this analysis of PfLDH known catalytic side, namely Asp155, Arg158, and His182. Tertiary structure prediction was performed using some software such as HH-suite, Clustal, SWISS-Model, and I-TASSER. Tertiary structure prediction performed with templates from Human LDH (protein 1I0Z) showed superimposed results with two unique differences, namely the addition of five amino acids to PfLDH residue 90–94 in the region of substrate specificity and conformational differences in PfLDH residue 210–215 in the antigenic loop. The next step is to carry out the expression of the PfLDH coding gene on E. coli BL21 as a biomarker for the RDT to be developed. First, a restriction analysis was performed to confirm the recombinant plasmid pET-30a-PfLDH using NcoI and XhoI enzymes. The presence of DNA fragments measuring 5210 bp and 954 bp showed the recombinant plasmid pET-30a-PfLDH was as expected. The transformation of E. coli BL21 with pET-30a-PfLDH was carried out using the heatshock method. Then, colony PCR was performed to screen the E. coli BL21 / pET-30a-PfLDH. Colony PCR performed using the pairing primers of PfLDHF (NcoI) and PfLDHR (XhoI) and yielded 12 cloned DNA fragment size of 952 bp. Furthermore, PfLDH expression was carried out at 30 ? with IPTG induction of 0.5 mM for 3 hours. From the analysis of the expression results using SDS-PAGE, it was concluded that PfLDH was successfully expressed as a protein with a molecular weight of 33 kDa.