Purification of the membrane-form variant surface glycoprotein of Trypanosoma brucei.

The membrane-form variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of African trypanosomes by a diacylglycerol residue. On cell rupture the anchor is rapidly cleaved by an endogenous phospholipase C. A purification procedure is described which results in native mfVSG devoid of lipase activity. A total membrane fraction is prepared in the presence of the SH-inhibitor p-chloromercuribenzenesulphonic acid (pCMBS). Membrane proteins are solubilized in the presence of pCMBS and the detergent Zwittergent 3-12, conditions which inhibit the activity of the phospholipase. mfVSG is then purified by successive chromatography on rabbit anti-VSG affinity and cation-exchange columns (25% yield). The isolated protein is electrophoretically pure and partitions into the detergent phase on Triton X-114 phase separation, proving that it retains the diacylglycerol anchor.