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Use of split-dihydrofolate reductase for the detection of protein-protein interactions and simultaneous selection of multiple plasmids in Plasmodium falciparum

Levray, YS; Berhe, AD; Osborne, AR; (2020) Use of split-dihydrofolate reductase for the detection of protein-protein interactions and simultaneous selection of multiple plasmids in Plasmodium falciparum. Molecular and Biochemical Parasitology , 238 , Article 111292. 10.1016/j.molbiopara.2020.111292. Green open access

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Abstract

Defining protein-protein interactions is fundamental to the understanding of gene function. Protein-fragment complementation assays have been used for the analysis of protein-protein interactions in various organisms. The split-dihydrofolate reductase (DHFR) protein-fragment complementation assay utilises two complementary fragments of the enzyme fused to a pair of potentially interacting proteins. If these proteins interact, the DHFR fragments associate, fold into their native structure, reconstitute their function and confer resistance to antifolate drugs. We show that murine DHFR fragments fused to interacting proteins reconstitute a functional enzyme and confer resistance to the antifolate drug WR99210 in Plasmodium falciparum. These data demonstrate that the split-DHFR method can be used to detect in vivo protein-protein interactions in the parasite. Additionally, we show that split-DHFR fragments can be used as selection markers, permitting simultaneous selection of two plasmids in the presence of a single antifolate drug. Taken together, these experiments show that split-DHFR represents a valuable tool for the characterisation of Plasmodium protein function and genetic manipulation of the parasite.

Type: Article
Title: Use of split-dihydrofolate reductase for the detection of protein-protein interactions and simultaneous selection of multiple plasmids in Plasmodium falciparum
Location: Netherlands
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.molbiopara.2020.111292
Publisher version: https://doi.org/10.1016/j.molbiopara.2020.111292
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Malaria, Plasmodium transfection, co-transfection, protein-fragment complementation assay, selection marker, split-dihydrofolate reductase
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: https://discovery.ucl.ac.uk/id/eprint/10101615
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