TY - JOUR
T1 - Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei
T2 - expression in Escherichia coli
AU - Mensa-Wilmot, Kojo
AU - Englund, Paul T.
N1 - Funding Information:
This work was supported by NIH grant AI21334 (to PTE) and by grants from the Rockefeller and MacArthur Foundations. KMW was a special postdoctoral fellow of the Rockefeller Foundation.
PY - 1992/12
Y1 - 1992/12
N2 - 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).
AB - 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).
KW - Expression in Escherichia coli
KW - Glycosyl phosphatidylinositol-specific phospholipase C
KW - Trypanosoma brucei
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U2 - 10.1016/0166-6851(92)90180-R
DO - 10.1016/0166-6851(92)90180-R
M3 - Article
C2 - 1362451
AN - SCOPUS:0026621595
SN - 0166-6851
VL - 56
SP - 311
EP - 321
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 2
ER -