Expression and Purification PfLDH in E. coli
Nursyifa Eva, Sugiyo Ignatius Yovin, Natalia Dessy, Masduki Fifi Fitriyah

Institut Teknologi Bandung
Jalan Ganesha 10, Bandung 40132, Indonesia

Abstract

Malaria is a disease caused by the Plasmodium parasite which is spread by the female Anopheles mosquito. Most malaria cases found in Indonesia are caused by infection with P. falciparum and P. vivax. One way to control malaria is malaria diagnosis. Rapid and accurate diagnosis of malaria is essential for effective control of the disease. Commercially available malaria RDTs in Indonesia 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 aims of this study were to analyze B cell epitope linear structure and conformation in silico, PfLDH protein expression, purification of PfLDH recombinant protein and characterization of PfLDH recombinant protein using RDT. The stages of the research include (i) predicting linear structure B cell epitope with the Bepipred method for early prediction of B cell epitope, Emini Surface Accessibility to determine the epitope position, Chou and Fasman to determine the beta turn structure, Predictability of Karplus and Schulz Flexibility to determine the ability and Parker to determine the hydrophilic nature of the antigen, while for the prediction of B cell epitope conformational structure was carried out by the DiscoTope method to determine the prediction of B cell epitope in the tertiary structure (ii) to express PfLDH recombinant protein (iii) to purify PfLDH recombinant protein. Based on the analysis of B cell epitope linear structure and conformation, the potential epitope candidate is APGKSDKEWNRDDL with an amino sequence of 85-98 with a length of 13 mer, where the candidate epitope is on the surface, is hydrophilic, flexible, and antigenic. The expression of recombinant PfLDH with IPTG 0.3 mM at 37oC was successfully carried 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(DE3) and Escherichia coli BL21(DE3)- RIPL bacterial cells has been successfully carried out and confirmed by SDS PAGE analysis which shows of thick fragments in the protein with a molecular weight of around 37 kDa. Characterization with RDT has been successfully carried out with the discovery of two bands in the control area and tests that confirm that there is a specificity between the antigens on the recombinant protein. This research is expected to contribute to the development of RDT malaria kits in Indonesia.

Keywords: PfLDH, E. coli

Topic: LIFE SCIENCES