Rapid preparative scale purification of myristylated variant surface glycoproteins from African trypanosomes.
A rapid method for the preparative-scale purification of variant surface glycoproteins (VSG) from African trypanosomes has been developed to investigate the recently described myristylated form of this molecule. This high-performance liquid chromatography (HPLC) procedure gives high yields and can be achieved in as little as 1 h from the preparation of a cell lysate in 0.1% trifluoroacetic acid (TFA). Solubilization of myristylated VSG in 0.1% TFA was shown to be much more effective than in neutral or zwitterionic detergents. Surface iodination of bloodstream trypanosomes and subsequent lysis in TFA showed that no proteolytic degradation of VSG occurred during solubilization and purification. Biosynthetic labelling of trypanosomes with [3H]myristic acid and purification of VSG by the described method, demonstrated that the VSG possessed covalently linked fatty acid and can therefore be defined as the membrane form of VSG. Evidence was provided that the myristic acid was covalently associated with the C-terminal portion of the VSG via a hydroxyester bond. Solubilization of the myristylated VSG by 0.1% TFA could be interpreted to indicate that the fatty acid was not embedded in the trypanosome membrane.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D006868:Hydrolysis; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009226:Myristates / Q000378:metabolism*; D009227:Myristic Acids / Q000378:metabolism*; D014347:Trypanosoma brucei gambiense / Q000032:analysis*; D014357:Trypsin
D006023:Glycoproteins; D008565:Membrane Proteins; D009226:Myristates; D009227:Myristic Acids; D014357:Trypsin