Overexpression, purification and localization of apoptosis related protein from Plasmodium falciparum.
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.
Guha
Mithu
M
Drug Target Discovery and Development Division, Central Drug Research Institute, Chatter Manzil Palace, Mahatma Gandhi Marg, Lucknow, Uttar Pradesh 226001, India.
Choubey
Vinay
V
Maity
Pallab
P
Kumar
Sanjay
S
Shrivastava
Kumkum
K
Puri
Sunil K
SK
Bandyopadhyay
Uday
U
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression / Q000502:physiology*; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000737:chemistry*; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000953:Antigens, Protozoan; C403009:PfARP protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins