Strategy to tag Actin II in Plasmodium berghei
Abstract: Malaria is a disease that is caused by parasite called Plasmodium spp. and trasmitted by female Anopheles mosquitoes to the host. The disease has great impact around the world and there are half a million deaths and several hundred million infections every year. Studies revealed that there are two actin isoforms in the parasite, actin I and actin II. Absence of actin II has severe effect on the development of the parasite in the mosquito but the molecular function is still unknown. Identification of interacting proteins is of great importance to understand further the function of the protein. To achieve this goal actin II has to be enriched and this required a tagged version of the protein. In this project purification of the protein was to be achieved through biotinylation. In this method the protein of interest is biotinylated by BirA ligase in the cell and is then purified by , streptavidin. The project involved transfection of vector for Plasmodium berghei, containing the BirA gene and a stage-specific promoter (cdpk4). The construct was integrated in the chromosomal locus Sil6 and introduced to wild-type and actin II knock out parasites. Genotyping by PCR revealed integration of the insert in wild type parasites and phenotypic anaylsis showed no difference between BirA wild type and wild type control parasites. The expression of the BirA ligase in the parasite was investigated with Western blot but no signal was detected.
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