[24] Purification of TRAPP from Saccharomyces cerevisiae and identification of its mammalian counterpart

ABSTRACT This chapter discusses the purification of TRAPP from Saccharomyces cerevisiae and identification of its mammalian counterpart. Primarily construction of Bet3p-protein A fusion used to purify TRAPP is discussed. TRAPP is initially purified using a strain in which the sole copy of Bet3p is tagged at its carboxy terminus with three c-myc epitopes. The Bet3p-associated proteins are then purified from this strain by precipitating the complex from a lysate. This is done by incubating a clarified detergent extract with affinity purified anti-c-myc antibody bound to Affi-Gel. The bound protein is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This affinity purification led to the identity of several TRAPP subunits. In addition, large-scale purification of TRAPP and identification of TRAPP subunits, small-scale precipitation of TRAPP, and identification of mammalian TRAPP from HeLa lysates are described.