Abstract
Analysis of gene function in Plasmodium falciparum, the most important human malaria parasite, is restricted by the lack of robust and simple reverse genetic tools. Approaches to manipulate protein levels post-translationally are powerful tools to study protein-off effects especially in the haploid malaria parasite where genetic knockouts of essential genes are lethal. We investigated if the auxin-inducible degron system is functional in P. falciparum and found that degron-tagged yellow fluorescent protein levels were efficiently reduced upon addition of auxin which otherwise had no effect on parasite viability. The genetic components required in this conditional approach were co-expressed in P. falciparum by applying the small peptide 2A. 2A is a self-processing peptide from Foot-And-Mouth Disease virus that allows the whole conditional system to be accommodated on a single plasmid vector and ensures stoichiometric expression levels.
Highlights
Research on Plasmodium falciparum, the most important human parasitic pathogen, is hampered by the lack of simple approaches to study the function of parasite proteins, potential drug and vaccine targets
To test its applicability in coexpression of two and more genes in malaria research, we chose a 24 amino acid long Foot-And-Mouth Disease virus (FMDV) 2A sequence which has been optimized for high cleavage activity and has been shown to result in equivalent protein levels [5]
After transfection with pHGB_R2Y2B, transgenic 3D7_R2Y2B parasites were selected on 5 mg/ml blasticidin S. Those parasites were able to grow under blasticidin pressure where 2Amediated co-translational cleavage led to an individual blasticidin S-deaminase protein conferring drug resistance
Summary
Research on Plasmodium falciparum, the most important human parasitic pathogen, is hampered by the lack of simple approaches to study the function of parasite proteins, potential drug and vaccine targets. Dual or triple vector transfection strategies are usually used This approach further lowers the already poor transfection efficiency in P. falciparum resulting in stochastic co-expression in single cells as well as unpredictable stoichiometry of protein concentration [3]. Novel approach employs the small peptide 2A (,20 amino acids) from the Foot-And-Mouth Disease virus (FMDV) in polycistronic expression vectors [4]. This element - when cloned in between two genes in a single open reading frame - results in individual proteins of almost equimolar quantities by a co-translational ribosome ‘skipping’ mechanism [5]. 2A technology may improve conditional gene expression systems that generally depend on co-expression of multiple proteins in individual cells
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
