title: Systematic analysis of FKBP inducible degradation domain tagging strategies for the human malaria parasite Plasmodium falciparum.
abstract: Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.
authors: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.
authors: Mauro Ferreira
authors: MF
authors: de Azevedo
authors: Paul R
authors: PR
authors: Gilson
authors: Heloisa B
authors: HB
authors: Gabriel
authors: Roseli F
authors: RF
authors: Simões
authors: Fiona
authors: F
authors: Angrisano
authors: Jacob
authors: J
authors: Baum
authors: Brendan S
authors: BS
authors: Crabb
authors: Gerhard
authors: G
authors: Wunderlich
mesh_terms: D000818:Animals; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008288:Malaria / Q000469:parasitology*; D009154:Mutation; D010963:Plasmodium falciparum / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020543:Proteome; D015800:Protozoan Proteins / Q000737:chemistry; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D020543:Proteome; D015800:Protozoan Proteins; C050256:ring-infected erythrocyte surface antigen (RESA), Plasmodium falciparum; D049452:Green Fluorescent Proteins; D022021:Tacrolimus Binding Proteins