EXPRESSION AND PURIFICATION OF PLASMODIUM FALCIPARUM LACTATE DEHYDROGENASE (PFLDH) IN ESCHERICHIA COLI

• Main Author: Nursyifa, Eva
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/68877
• Institution: Institut Teknologi Bandung

Malaria is a disease caused by the Plasmodium parasite which is transmiteed by the female Anopheles mosquito. Plasmodium that infect humans are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. Malaria cases that are mostly found in Indonesia are caused by infection with P. falciparum and P. vivax. Cases of infection in Indonesia are still high, especially in Eastern Indonesia. Rapid and accurate diagnosis of malaria is essential for effective control of the disease. Immunochromatography-based rapid diagnostic test (RDT) which is economical, simple to perform, and gives results in a relatively short time to assist in the effective management of malaria. Commercially available malaria RDT are still being imported. Therefore, efforts are needed to produce malaria RDT independently. One of the biomarkers used in malaria RDT is pLDH (Plasmodium lactate dehydrogenase). The production and accumulation stage of all infected human can be used to indicate parasite survival, which is correlated with the number of parasites present in the plasma of infected patients. In this study, the gene encoding LDH was obtained from the P. falciparum genome which has high homology with the other four Plasmodium species so that it can be used for general Plasmodium detection. The purpose of this study was to express which is biomarker in silico analysis of PfLDH protein and PfLDH protein expression used as a component of malaria RDT. The stages of the research to include, (i)PfLDH predicting epitopes using the Bepipred, Emini Surface Accessibility, Chou Fasman, Karplus and Schulz Flexibility Prediction and Parker methods (ii) transforming pET16b-PfLDH to recombinant plasmid into strain to Escherichia coli host strains, namely the BL21 strain (D3), BL21-CodonPlus (DE3)-RIPL (iii) expressed PfLDH recombinant protein and (iv) characterized PfLDH protein with SDS PAGE. Based on the epitope analysis, it was found that the potential epitope candidates were epitopes with sequence SNTYDDLAG and FTKAPGKSDKEWNR. The express recombinant PfLDH with IPTG 0.3 mM at 37oC was successcarried out and confirmed by SDS-PAGE analysis, which showed the presence of protein fragments with a molecular weight of about 37 kDa. Purification of PfLDH recombinant protein in Escherichia coli BL21 bacterial host cells has been successfully carried out and confirmed by SDS PAGE analysis which shows the presence of a thick fragment in the protein with a molecular weight of around 37 kDa. This research is expected to contribute to the development of malaria RDT kits in Indonesia