Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli

Kojo Mensa-Wilmot, Paul T. Englund

Research output: Contribution to journalArticlepeer-review

Abstract

Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures. We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. We have purified large amounts of GPI-PLC from E. coli membranes, using a single step immunoaffinity technique. The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point. Recombinant GPI-PLC is a membrane enzyme; it associates with E. coli membranes and, like the T. brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments. The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate). The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a T. brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T. brucei brucei (ILTat 1.3).

Original languageEnglish (US)
Pages (from-to)311-321
Number of pages11
JournalMolecular and Biochemical Parasitology
Volume56
Issue number2
DOIs
StatePublished - Dec 1992

Keywords

  • Expression in Escherichia coli
  • Glycosyl phosphatidylinositol-specific phospholipase C
  • Trypanosoma brucei

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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