Use of the green fluorescent protein as a marker in transfected Leishmania

D. Sean Ha, James K. Schwarz, Salvatore J. Turco, Stephen M. Beverley

Research output: Contribution to journalArticle

208 Citations (Scopus)

Abstract

We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FAGS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FAGS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.

Original languageEnglish (US)
Pages (from-to)57-64
Number of pages8
JournalMolecular and Biochemical Parasitology
Volume77
Issue number1
DOIs
StatePublished - Apr 1996

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Leishmania
Green Fluorescent Proteins
Penicillin G Benzathine
Communicable Disease Control
Fluorescence
Proteins
Gene Targeting
Golgi Apparatus
Protein Transport
Codon
Endoplasmic Reticulum
Transfection
Polysaccharides
Parasites
Cell Count
Gene Expression
Mutation
DNA

Keywords

  • Fluorescent tag
  • GPI anchors
  • Glycolipid biosynthesis secretory pathway
  • Glycolipids
  • Lipophosphoglycan
  • Protozoan parasite

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

Cite this

Use of the green fluorescent protein as a marker in transfected Leishmania. / Sean Ha, D.; Schwarz, James K.; Turco, Salvatore J.; Beverley, Stephen M.

In: Molecular and Biochemical Parasitology, Vol. 77, No. 1, 04.1996, p. 57-64.

Research output: Contribution to journalArticle

Sean Ha, D. ; Schwarz, James K. ; Turco, Salvatore J. ; Beverley, Stephen M. / Use of the green fluorescent protein as a marker in transfected Leishmania. In: Molecular and Biochemical Parasitology. 1996 ; Vol. 77, No. 1. pp. 57-64.
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