Purification of the Glycosylphosphatidylinositol (GPI) transamidase. For purification of the GPI transamidase from the procyclic form, we extracted tagged Trypanosoma brucei (Tb)GPI8 from 10¹⁰ cells in 45 ml of TEN-NP buffer [50 mM Tris·HCl (pH 7.5)/5 mM EDTA/150 mM NaCl/Complete protease inhibitor cocktail (Roche Diagnostics)/1% Nonidet P-40] and removed cell debris by centrifugation at 18,800 ´ g for 20 min. We incubated 40 μl of a slurry of anti-FLAG M2 antibody beads (Sigma) with the extract for 6.5 h at 4° C, washed the beads five times with 1 ml of TEN-NP buffer, and eluted the bound proteins four times with 40 μl of FLAG elution buffer [50 mM Tris·HCl (pH 7.5)/150 mM NaCl/protease inhibitor cocktail/1% Nonidet P-40/1 mg/ml of FLAG peptide]. We combined the four eluates and mixed them with 340 μl of FLAG elution buffer and 30 μl of GST (for GST tag, Amersham Pharmacia Biotech) or TALON resin (for histidine affinity tag tag, Clonetech) slurry. After five washes in TEN-NP buffer, we eluted the transamidase complex three times with 30 μl of elution buffer [for GST tag, 20 mM Tris·HCl (pH 9.5)/150 mM NaCl/20 mM reduced glutathione; for HAT tag, 20 mM Tris·HCl (pH 8.0)/100 mM NaCl/100 mM imidazole]. An aliquot of the samples equivalent to 10⁹ cells instead was eluted with SDS/PAGE sample buffer with or without 2-mercaptoethanol and subjected to 10–20% gradient SDS/PAGE gel. After electrophoresis, the gel was silver-stained for visualization. The remaining samples were used for SDS/PAGE and blotting to poly(vinylidene difluoride) membrane to determine N-terminal amino acid sequences by protein sequencer (494, Applied Biosystems).

GPI transamidase of the bloodstream form-expressing TbGPI8-FLAG-GST was isolated in a similar way. For this, 5 ´ 10⁹ cells were obtained from blood of infected Sprague–Dawley rats with chromatography on DEAE-Sephacel (Sigma) columns (1).

1. Lanham, S. M. (1968) Nature 218, 1273–1274.