A new method for the rapid purification of both the membrane-bound and released forms of the variant surface glycoprotein from Trypanosoma brucei.
A simple new technique was developed for the rapid purification of either the membrane-bound or the released forms of the variant surface glycoprotein of Trypanosoma brucei in high yield. Whole cells were used as the source of the membrane-bound form, and the supernatant of benzyl alcohol-treated cells was used as the source of the released form. The technique was based on extraction of the acid-treated protein into chloroform/methanol, followed by selective re-partition into aqueous salt solution. The yield of purified protein was found to be dependent critically on a low pH during the extraction/re-partition stages. This finding and the ability to cycle the protein repeatedly through organic and aqueous phases in a strictly pH-dependent manner suggested that the protein could undergo fully reversible denaturation/renaturation only while in an extensively protonated form. The yield was independent of the polarity of the organic phase and the protein concentration over a wide range. After purification, both forms retain their ability to react with specific antibody raised against the authentic native protein purified by conventional means. The amino acid composition and the identity of the N-terminal amino acid was the same for both forms of the protein. In addition, both forms had blocked C-terminal residues. There were determined to be 1.13 X 10(7) copies of the variant surface glycoprotein per cell.
D000596:Amino Acids / Q000032:analysis; D002725:Chloroform; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000302:isolation & purification*; D000432:Methanol; D014346:Trypanosoma brucei brucei / Q000032:analysis*; D014643:Variant Surface Glycoproteins, Trypanosoma
D000596:Amino Acids; D006023:Glycoproteins; D008565:Membrane Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D002725:Chloroform; D000432:Methanol