2A and the auxin-based degron system facilitate control of protein levels in Plasmodium falciparum

PLoS One. 2013 Nov 13;8(11):e78661. doi: 10.1371/journal.pone.0078661. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism
  • Cysteine Endopeptidases / biosynthesis
  • Cysteine Endopeptidases / genetics
  • HeLa Cells
  • Humans
  • Indoleacetic Acids / pharmacology*
  • Inhibitory Concentration 50
  • Luminescent Proteins / metabolism
  • Plasmodium falciparum / drug effects
  • Plasmodium falciparum / metabolism*
  • Proteolysis / drug effects*
  • Protozoan Proteins / metabolism*
  • Transcriptional Activation
  • Viral Proteins / biosynthesis
  • Viral Proteins / genetics

Substances

  • Bacterial Proteins
  • Indoleacetic Acids
  • Luminescent Proteins
  • Protozoan Proteins
  • Viral Proteins
  • yellow fluorescent protein, Bacteria
  • Cysteine Endopeptidases

Grant support

This work was supported by the scientific budget of the Institute of Tropical Medicine, University Tübingen, Wilhelmstraße 27, D-72074 Tübingen, Germany. The authors acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tübingen University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.