Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli.
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).
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD.
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Mensa-Wilmot
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Englund
D000818:Animals; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D005971:Glutamates; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010738:Type C Phospholipases