Purification of components of the translation elongation factor complex of Plasmodium falciparum by tandem affinity purification.
Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1beta harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1beta-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1beta, the G-protein PfEF-1alpha, and two new proteins that we named PfEF-1gamma and PfEF-1delta based on their homology to other eukaryotic gamma and delta translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1beta. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.
Takebe
Sachiko
S
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3301, USA.
Witola
William Harold
WH
Schimanski
Bernd
B
Günzl
Arthur
A
Ben Mamoun
Choukri
C
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D017125:Genes, Protozoan; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000302:isolation & purification*; D010957:Plasmids; D010963:Plasmodium falciparum / Q000737:chemistry*; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011995:Recombination, Genetic
D046912:Multiprotein Complexes; D010445:Peptide Elongation Factors; D015800:Protozoan Proteins