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Updated: Jul 18, 2026
HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins
Published on: June 10, 2015
Overexpression, purification and localization of apoptosis related protein from Plasmodium falciparum
Mithu Guha1, Vinay Choubey, Pallab Maity
1Drug Target Discovery and Development Division, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg, Lucknow, Uttar Pradesh 226001, India.
Abstract:
A growing body of evidence has ascertained that apoptosis is not only restricted to metazoans but also exists in unicellular parasites. In Plasmodium falciparum, the presence of a putative gene having sequence homology with apoptosis related protein (PfARP) (Gene ID PFI0450c) has raised enormous interest to unravel the function of this unique protein in cell death of malaria parasite. To characterize this protein, the PfARP gene has been amplified from the P. falciparum transcriptome by RT-PCR and the amplified gene has been successfully cloned, over-expressed and purified to homogeneity. The purified PfARP exhibits minimum subunit MW of approximately 24kDa as evident from SDS-PAGE. CD analysis reveals that the alpha and beta content of the recombinant PfARP are 61% and 15%, respectively. Semiquantitative RT-PCR analysis indicates the expression of PfARP at both metabolically less active ring and highly active trophozoite stages of malaria parasite. Immunofluorescence microscopy further supports that PfARP expresses stage specifically with the highest expression at trophozite stage and very little in the schizont stage. PfARP is a cytosolic protein as evident from immunofluorescence microscopy. The role of this protein in P. falciparum cell death and stage progression is not yet known. The identification, purification and characterization would certainly be a step to initiate work on this protein to evaluate its role in P. falciparum growth, multiplication and stage progression.
Insights
Apoptosis occurs in unicellular parasites like Plasmodium falciparum. Researchers identified and purified a novel apoptosis-related protein (PfARP), a crucial step to understanding its role in malaria parasite cell death.
Area of Science:
- Parasitology
- Molecular Biology
- Cell Biology
Background:
- Apoptosis, a programmed cell death, is increasingly recognized in unicellular organisms, including parasites.
- The malaria parasite, Plasmodium falciparum, possesses a putative apoptosis-related protein (PfARP) (Gene ID PFI0450c) of unknown function.
- Understanding PfARP's role is vital for novel malaria control strategies.
Purpose of the Study:
- To characterize the Plasmodium falciparum apoptosis-related protein (PfARP).
- To investigate the expression patterns and localization of PfARP within the malaria parasite.
- To lay the groundwork for future studies on PfARP's function in parasite cell death and development.
Main Methods:
- Gene amplification via RT-PCR from P. falciparum transcriptome.
- Cloning, over-expression, and purification of recombinant PfARP.
- SDS-PAGE for molecular weight determination.
- Circular Dichroism (CD) analysis for secondary structure assessment.
- Semi-quantitative RT-PCR and immunofluorescence microscopy for expression analysis and localization.
Main Results:
- PfARP was successfully cloned, over-expressed, and purified, with a subunit molecular weight of approximately 24kDa.
- CD analysis indicated significant alpha-helical (61%) and beta-sheet (15%) content.
- PfARP expression was detected in ring and trophozoite stages, with highest levels in trophozoites.
- Immunofluorescence microscopy confirmed PfARP as a cytosolic protein, predominantly expressed in the trophozoite stage.
Conclusions:
- The study successfully identified, purified, and characterized PfARP, a putative apoptosis-related protein in Plasmodium falciparum.
- PfARP exhibits stage-specific expression, primarily during the trophozoite stage, and is localized in the cytosol.
- This foundational work provides essential data for future investigations into PfARP's role in malaria parasite cell death, growth, and stage progression.
